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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 (T– 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, P1P2P3cand 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, Nigeriawere 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.



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 Temperature                                                                                                    27 Soil Characteristics                                                                                         28 pH                                                                                                                   28 Oxygen                                                                                                           29 Nutrient availability                                                                                        30 Microorganisms number and catabolism evolution                                         30 Consortium of microorganisms                                                                       31 Bioavailability                                                                                                 32 Contaminant characteristics                                                                            33 Toxicity of end-products                                                                              34 Moisture                                                                                                        35 Organic matter                                                                                              35 Oil surface and concentration                                                                       35 Salinity                                                                                                          36

2.6       Bioremediation                                                                                               36

2.6.1    Bioremediation techniques                                                                             38 Biological analysis                                                                                         39 Soil respirometry                                                                                          39  Luminescence techniques                                                                           39 Dehydrogenase activity                                                                               40 Chemical analysis                                                                                           40 Gas chromatography (GC)                                                                           40 Gas chromatography/mass spectroscopy (GC/MS)                                     41 Gas chromatography/flame ionization detection (GC/FID)                        41 Fluorescence analysis                                                                                   41 Use of internal petroleum biomarkers                                                          42 Total petroleum hydrocarbon/infrared spectroscopy (TPH/IRS) –

                            Total petroleum hydrocarbon/gas chromatography (TPH/GC)                  42   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



5.1       Conclusion                                                                                                      119

5.2        Recommendations                                                                                         119








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







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









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.


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


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.


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.



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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