BIOCHEMICAL AND METAGENOMIC STUDIES OF PETROLEUM CONTAMINATED

SOILS OF OGONILAND, RIVERS STATE, NIGERIA

Ime Etim NDEKHEDEHE

BSc (Hons.) (Uyo, MSc (Uyo)
16/PG/BMS/BC/PhD/004

SUPERVISOR: CO-SUPERVISORS:
Dr. (Mrs) Justin I.R. Udotong Dr. Oboso E. Etim
Dr. Queensley A. Adesuwa.

JUNE 2023

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BIOCHEMICAL AND METAGENOMIC STUDIES OF PETROLEUM CONTAMINATED
SOILS OF OGONILAND, RIVERS STATE, NIGERIA

Ime Etim NDEKHEDEHE

BSc (Hons.) (Uyo, MSc (Uyo)
16/PG/BMS/BC/PhD/004

A THESIS IN THE DEPARTMENT OF BIOCHEMISTRY,

FACULTY OF SCIENCE

SUBMITTED TO

THE POSTGRADUATE SCHOOL, UNIVERSITY OF UYO, UYO, AKWA IBOM STATE,

NIGERIA, IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF
DOCTOR OF PHILOSOPHY (PhD) DEGREE IN BIOCHEMICAL TOXICOLOGY

JUNE 2023
DECLARATION
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I, Ime Etim NDEKHEDEHE, hereby declare that this doctoral thesis entitled “Biochemical and

Metagenomic Studies of Petroleum Contaminated Soils of Ogoniland, Rivers State, Nigeria” was

written by me and that it is the correct record of my own research work. It has not been presented

for a degree in another institution. I will not present it, or cause it to be presented, for a degree in

another institution. All sources of information have been appropriately acknowledged using

references and other acceptable methods.

Signed………………………. Date……………….
Ime Etim NDEKHEDEHE

CERTIFICATION
iii
This thesis titled “Biochemical and Metagenomic Studies of Petroleum Contaminated Soils of

Ogoniland, Rivers State, Nigeria” by Ime Etim NDEKHEDEHE (16/PG/BMS/BC/PhD/004) meets

the regulations governing the award of the PhD degree of the University of Uyo. The work has

made original contribution to knowledge. It should be submitted to the Postgraduate School for

approval.

(a) Supervisor

Signature …………………………………………….. Date………………………..

Name: Asso. Prof. Justina I. R. Udotong Rank………………………..

Department/Faculty:………………………………………………………………………

(b) Co-Supervisor

Signature …………………………………………….. Date………………………..

Name: Dr. Oboso E. Etim Rank………………………..

Department/Faculty:……………………………………………………………………….

(c) Internal Examiner

Signature …………………………………………….. Date………………………..

Name: Dr. Olajide J. Akinjogunla Rank………………………..

Department/Faculty:……………………………………………………………………..

(d) Head of Department and Chief Examiner

Signature …………………………………………….. Date………………………..

Name: Dr. Jessie I. Ndem Rank………………………..

Department/Faculty:………………………………………………………………………

(e) External Examiner

Signature …………………………………………….. Date………………………..

Name: Prof. Donatus Chuka Belonwu Rank………………………..

Department/Faculty:………………………………………………………………………..

DEDICATION
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I dedicate this work to my juicy husband, Pastor Asuquo Etim Ndekhedehe

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 ACKNOWLEDGEMENTS

I wish to express my profound gratitude to my supervisor Dr. (Mrs.) Justina I. R. Udotong, for her

love, contributions and support during the course of the research work. Also my sincere gratitude

goes to my Co-supervisors Dr. Oboso E. Etim, and Dr. Queensley A. Adesuwa whose advice has

added value to my work. This work cannot be complete without mentioning my gratitude to Prof.

Ime Udotong, Prof. J.P. Essien, Prof. Tatfeng Maribeau, Prof. Edet Akpanyung, Prof. Enomfon

Akpan, Dr. Jessie Ndem, Dr. Akin .O, Dr. Shaibu .S, Dr. Efosa Ewere, Dr. Amana, Dr. Fatunla

Opeyemi, Dr. Innocent Edagha, and Dr. Ebe. whose knowledgeable advice has given me the

strength to complete this research.

Many thanks to senior technologist Mrs. Christie Udosen of the Department of Microbiology,

University of Uyo, and Mr. Adenekan of the Department of Biochemistry, University of Lagos, for

their help towards this work.

My intense gratitude to my juicy husband, Pastor Asuquo Etim Ndekhedehe, my lovely angelic

children Deborah, Esther, Lydia and Janet for their love, support, prayers and patience towards my

academics.

Also want to appreciate my sisters, Mrs. Janet Ogba, Mrs. Ikwo Oka, Mrs. Affiong Obiam; to my

brother in-laws, Mr. Ukeje Ogba, Mr. Asuquo Ndekhedehe and Mr. Emeka Obiam for all your

care. God bless you all. Also appreciate my father Elder Udo John Udo, father in-law Elder

Asuquo Ndekhedehe and mother in-law Elder (Mrs.) Cecilia Ndekhedehe, for motivating me and

also Elder (Mrs.) Lucy Ndekhedehe and Mrs. Abasiumoh for being there for me.

To all my friends and loved once especially Dr. Ekpo Ndifreke, Mr Victor, Leonard, Nathaniel,

Segun, Paul, Patience, Ini, Victoria, and Livinus. I want to say a very big thank you.

My gratitude to Pastor and Mrs. Ilori, Pastor and Mrs. Joshua, Pastor and Mrs. Mathew, Pastor and
Mrs. Emeka, Pastor and Mrs. Henry, Pastor and Mrs. Okonzua, Pastor and Mrs. Adeyanju and
Pastor Titus for their prayers. Finally, I am grateful to God Almighty whose grace has been
sufficient for me to complete my research work.

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 ABSTRACT

The effect of oil spillage from oil exploration especially in petroleum-producing areas is alarming.
It is because of this challenge that the biochemical activities of hydrocarbon-degrading bacteria
(HDB) and eubacterial diversity in polluted soils at B. Dere in Gokana LGA of Rivers State,
Nigeria was investigated using standard analytical techniques and molecular approach standard
analytical techniques and molecular approach analytical techniques and molecular approach. The
biodegradation products from the consortia of HDB were closely monitored by gas
chromatography-mass spectrometry (GC-MS) technique. Results obtained for the physicochemical
parameters were: temperature, 27 to 28oC, pH, 6.2 to 6.3, electrical conductivity, 301.5 to 306.4
u/scm, moisture content, 10.55 to 11.61, chloride, 37.91 to 40.0 mg/L, nitrate, 6.00 to 6.10 mg/L,
nitrite, 2.01 to 2.05 mg/L, phosphate, 7.13 to7.15 mg/L, sulphate, 83.58 to 84.06 mg/L, organic
matter, 70.95 to 71 %,THC, 26.68 to 29.09, TOC, 18.72 to18.90 %, silt, 7 to 8, clay, 15-16, and
sand, 53. The soil sample produced a significantly (p < 0.001) higher inhibition of DPPH radical,
in concentration-dependent manner, relative to the standard (ascorbic acid). The HDB isolates in
the contaminated soil were: Pseudomonas sp., Salmonella, Citrobacter sp., Bacillus megaterium
and Bacillus subtilis. Enzymatic analysis of the HDB isolates revealed the presence of catalase,
urease, succinate dehydrogenase, peroxidase and protease. Bacillus subtilis recorded the highest
catalase activity (668.16±79.92 μmol/mL/min). Urease activity was highest in Bacillus subtilis
(0.25 ± 0.00 mg/g/min). Succinate dehydrogenase activity was highest in Pseudomonas sp. (5.67 ±
0.00 μmol/mL/min). Peroxidase activity was highest in Bacillus megaterium (76.04 ± 0.00
μmol/mL/min). Bacillus megaterium recorded the highest (91.08 ± 0.68 units/mg) in protease
activities. Catalase activity was significantly (p < 0.001) higher than the activities of other enzymes
assayed. Culture-dependent method revealed the presence of 16 bacteria (Bacillus subtilis,
Micrococcus sp., Bacilllus cereus, Nitrobacter sp., Citrobacter sp., Pseudomonas putida, Vibrio
cholerae, Nocardia sp., Shigella sp., Salmonella sp., Escherichia coli, Staphylococcus albus,
Clostridium sp., Pseudomonas aeruginosa, Nitrosomonas sp. and Bacillus megaterium) in
contaminated soil. Metagenomics method revealed bacteria as 100% in the domain,
Actinobacteriota was dominant in phyla and Bacillus was dominant in the genera. GC-MS results
revealed that Bacillus subtilis significantly degraded the heavy crude oil to carboxylic acid with
smaller molecular mass which was closely followed by Pseudomonas aeruginosa. Most of the
PAHs like naphthalene and anthracene in the control sample were biodegraded to lighter
polycyclic hydrocarbons. Essentially, the hydrocarbon degrading bacteria demonstrated the ability
as potent biodegraders and survivors in oil-contaminated soils, but also utilize hydrocarbons for
growth and energy source.

[400 Words]*

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 TABLE OF CONTENT

Chapter Title Page

Front Cover
Cover Page i
Title Page ii
DECLARATION iii
CERTIFICATION iv
DEDICATION v
ACKNOWLEDGEMENTS vi
ABSTRACT vii
TABLE OF CONTENTS viii
LIST OF TABLES xiii
LIST OF FIGURES xiv
LIST OF APPENDICES xv
LIST OF ABBREVIATIONS AND ACRONYMS xvi

CHAPTER ONE: INTRODUCTION

1.1 Background of the Study 1-3

1.2 Statement of Problem 3-4
1.3 Justification of the Study 4
1.4 Objective of the Study 6

1.4.1 General Objective of the Study 6

1.4.2 Specific Objectives of the Study 5

1.5 Scope of the study 6

CHAPTER TWO: REVIEW OF RELATED LITERATURE

2.1 The Impact of Oil Spill 7
2.1.1 Impact of Environmental Health Problems in Ogoniland 8
2.1.2 Effect of Oil Spill on Human Health in Ogoniland 9
2.1.3 Effects of Oil Spills on Agricultural Production in Ogoniland 9-10
2.2 Hydrocarbons 10-11
2.2 Types of Hydrocarbon 11

2.2.2 Alkanes and Alkenes 11-12

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2.2.3 Aromatic Hydrocarbons 13-15
2.2.4 Hydrocarbon Biodegrative Microorganisms 15-16
2.2.4.1 Bacteria 16-17
2.2.4.2 Fungi 17-18
2.2.4.3 Yeast 18
2.2.4.4 Algae 18-19
2.3 Bioremediation 19-20
2.3.1 Factors Affecting Bioremediation 21-22
2.3.1.1 Biological Factors 22
2.3.1.2 Environmental Factors 22-25
2.4 Biocatalysts Involved in Bioremediation of Oil Polluted Soil Environment 25-28
2.4.1 Enzyme Classification 28
2.4.1.1 Microbial Oxidoreductases 29-30
2.4.1.2 Oxidases 30-31
2.4.1.3 Dehydrogenases 31-32
2.4.1.4 Laccases 33-34
2.4.1.5 Peroxidases 34-36
2.4.1.6 Proteases 37
2.4.1.7 Hydrolytic Enzymes 39-42

2.4.1.8 Cellulases 42-43

2.4.1.9 Proteases 43-44
2.5 Metagenomics: An Application Based Perspective 45-47
2.5.1 Molecular Method 48
2.7.1.1.1 Metagenomic DNA Extraction 48-49

2.5.1.2 Calculation of Concentration and Purity of Metagenomic DNA Extracts 49-50

2.5.1.3 Bioinformatics Method 50-51
2.5.1.4 Assembly 51
2.5.1.5 Binning 51
2.5.1.6 Sequence Analysis 51
2.5.1.7 Phylogenetic Analysis 51-52

CHAPTER THREE: METHODOLOGY

3.1 Materials 54
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3.1.1 Description of the Study Area 54
3.1.2 Study Site 54
3.2 Polluted Soil Collection and Pretreatment of Polluted Soils Samples 55
3.3 Analysis of Physico-chemical Parameters of polluted Soil Samples 56
3.3.1 Determination of in-situ Parameters of polluted Soil Samples 56
3.3.2 Determination of Chemical Parameters of polluted Soil Samples 56
3.3.2.1 Determination of Nitrate (NO3-) Content 56-57
3.3.2.2 Determination of Nitrite (NO2-) Content 57-58
3.3.2.3 Determination of Phosphate (PO42-) Content 58-
3.3.2.4 Determination of Sulphate (SO42-) Content 58-59
3.3.2.5 Determination of Total Organic Carbon 60
3.3.2.6 Determination of Total Hydrocarbon Content 60
3.3.2.7 Determination of Total Organic Carbon (TOC) 60-61
3.3.2.8 Determination of Chloride (Cl-) Content 61
3.4 Biochemical and Scavening Activity of HDB in the Polluted Soil Samples 62
3.4.1 Determination of Scavenging Activity of Hydrocarbon Degrading Bacteria using
DPPH (2,2-diphenyl – picrylhydrazyl) Scavenging Assay 62
3.4.2 Determination of Enzymatic Activity in Hydrocarbon Degrading Bacteria 62
3.4.2.1 Assay of Catalase 62-63
3.4.2.2 Assay of Peroxidase 63
3.4.2.3 Assay of Succinate Dehydrogenase 63-64
3.4.2.4 Assay of Urease (Amidohydrolase) 64
3.4.2.5 Assay of Protease 64
3.5 Microbiological Analysis of the Polluted Soil Samples 65
3.5.1 Analysis of Oil Polluted Soil Samples 65
3.5.1.1 Ten-Fold Serial Dilution 65
3.5.1.2 Estimation of Microbial Densities 65-66
3.5.1.3 Determination of Coliform Communities 66
3.5.1.4 Incubation of Culture Plates and Counts of Microbial Colonies 66
3.5.1.5 Purification and Maintenance of Microbial Isolates 67
3.5.1.6 Characterization and Identification of Microbial Isolates 67
3.5.2 Determination of Hydrocarbonoclastic Bacteria 67-72

3.6 Determination of Eubacterial Diversity of Hydrocarbon Polluted Soils in 73-75

B.Dere,Gokana LGA.
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3.7 Gas Chromotography-Mass Spectrometer (GC-MS) Analysis. 75-76
3.8 Statistical Analysis 76
CHAPTER FOUR: RESULTS AND DISCUSSION
4.1 Results 77
4.1.1 Physico-chemical Analysis 77-79
4.1.2 Scavenging Activity of Hydrocarbon Degrading Bacteria 84
4.1.3 Enzymatic Activity of Hydrocarbon Degrading Bacteria 86-87
4.1.4 Estimation of Microbial Densities using Culture-Dependent Methodology 88-89
4.1.4.1 Characterization (Biochemical Studies) and Identification of Microbial Isolates 99

4.1.4.2 Classification of Bacteria into Taxonomic Groups 99

4.1.5 Eubacterial Diversity Profile in Hydrocarbon Polluted Soils in B.Dere, Gokana LGA 100

4.1.6 GC-MS Analysis of Remediated Soil Sites by Consortium of Microorganisms 106

4.1.6.1 GC-MS of abiotic control sample of the crude oil 106

4.2 Discussion

CHAPTER FIVE: SUMMARY, CONCLUSION AND RECOMMENDATIONS

5.1 Summary 118
5.2 Conclusion 119-120
5.3 Scientific Implication 120
5.4 Contributions to Knowledge 120-121
5.5 Recommendations 121
5.6 Suggestion for Further Studies 121
LIST OF TABLES

TABLE TITLE PAGE

2.1: Environment threats of petroleum hydrocarbon contamination. 42

2.2: Microorganismshavingdegradationpotentialforphenolicsandbenzoates 48

2.3: Enzymes involved in biodegradation of petroleum hydrocarbons.

4.1: Physico-chemical properties of oil contaminated soil sample 83

4.2: Physico-chemical properties of non-contaminated soil sample 84

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4.3: Effects of Physico-chemical parameters on bioremediation 86

4.4: DPPH scavenging activity (% inhibition) of bacteria extract from 87

contaminated soil sample

4.5: DPPH scavenging activity (%inhibition) of bacteria extract from 89

non-contaminated soil sample (control)

4.6: Enzymes activity from Bacteria isolate (hydrocarbon degrading bacteria 89

that is involved in Bioremediation

4.7: Microbial densities of oil polluted soil sample 90

4.8: Microbial densities of non-polluted soil sample (control) 90

4.9: Characterization and identification of bacteria isolated from contaminated 91

soil using culture-dependent methods

4.10: Characterization and identification of bacteria isolated from non-contaminated site

(control) using culture-dependent techniques 92

4.11: Morphological characterization of fungal isolates from oil contaminated soil sample
using culture-dependent techniques. 93
4.12: Microbial densities of non-polluted soil sample (control). 93
4.13: Classification of bacteria isolates of oil polluted soil sample from culture-dependent
method into taxonomic levels. 95-96
4.14: Characterization and identification of bacteria isolated from non-contaminated site
(control) using culture-dependent techniques. 96-98
4.15: Morphological characterization of fungal isolates from oil contaminated
soil sample using culture-dependent techniques.
4.16: Morphological characterization of fungal isolates from non- contaminated soil

sample using culture-dependent techniques 98

4.17 Classification of bacteria isolates of oil polluted soil sample from culture-dependent

method into taxonomic levels. 99

4.18: Classification of the bioactive compounds that was abundant for biodegradation100

LIST OF FIGURES

FIGURE TITLE PAGE

1.1: Oil spillage in B.dere in Ogoniland 6

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2.1: Graph showing the levels of oil spill. 8

2.2: Biochemical pathway for protease hydrolysis 41

3.1: Description of sampling point in Gokana LGA Rivers State, Nigeria. 59

4.1: Scavenging activity of hydrocarbon degrading bacteria. 100

4.2: Catalase activities of hydrocarbon degrading bacterial 101

4.3: Urease activities of hydrocarbon degrading bacterial. 102

4.4: Succinate hydrogenase activities of hydrocarbon degrading 103

bacterial

4.5: Peroxidase activities of hydrocarbon degrading bacterial. 104

4.6: Protease activities of hydrocarbon degrading bacteria. 105

4.7: Phyla classification of oil polluted soil sample. 106

4.8: Classes classification of oil polluted soil sample. 107

4.9: Order classification of oil polluted soil sample. 108

4.10: Families classification of oil polluted soil sample. 109

4.11: Genera classification of oil polluted soil sample. 110

4.12: Phyla classification of bacteria non-contaminated soil sample. 111

4.13: Classes classification of non-contaminated soil sample. 112

4.14: Order classification of non-contaminated soil sample. 113

4.15: Families classification of non-contaminated soil sample. 114

4.16: Classification Rate by Taxonomic Level for bacteria 115

4.17: GC chromatogram of (A) bioremediated sample with Baccillus sp 116

4.18 GC chromatogram of (A) bioremediated sample with Streptococcus sp 117

4.19 GC chromatogram of (A) bioremediated sample with Serratia sp 118

Reference 122-142
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My Published Works and Expected Publications 143

Appendices 144-164

LIST OF ABBREVIATIONS AND ACRONYMS

Abbreviations Meaning

AC Actionmycetes count

ADP Adeine-5’ - diphosphate

APX Ascorbate peroxidase

ARP Arthromyces ramosus perxidase

ATP Adenine triphosphate

B.Dere Baranyowa dere

BLAST Basic Local Alignment Tool

BRPD Bleomycin resistance dioxygenase

BSA Bovine serum albumin

CA Centrimide agar

CBH Cellobiohydrolase

COSs Chito-oligosaccharides

CS Contaminated soil

CS1 Contaminated soil replicate 1;

CS2 Contaminated soil replicate 2;

DI Dionized

DPPH 2,2-Diphenyl-picrylhydrazyl
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EC Electrical conductivity

EC Enzyme commission

EDTA Ethylenediaminetetracetic acid

EG Englucanase

EMBA Eosine methylene blue agar

FCC Faecal coliform count

FDM Flavin-dependent monoxygenase

GC-MC Gas chromatography-mass spectrometer

G/c NAC N-Acetylglucosamine

HDB Hydrocarbon degrading bacteria

HUBC Hydrotolerate hydrocarbon utilizing bacteria consortium

LiP Lignin peroxides

LPS Lipopolysaccharides

MAC MacConkey agar

MnP Manganese peroxides

MOCs Multinational oil companies

MSA Manitol salt agar

MV-VP Methyl red-Voges Proskaures

NCS Non-contaminated soil

NCS 1 Non-contaminated soil replicate 1;

NCS 2 Non-contaminated soil replicate 2;

NGS Next generation sequencing

NR Nitrate reducers

OFDE Oxo-flavin degrading enzyme

OLC Overlapping layout consensus

OM Outer membrane
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PAHs Polycyclic aromatic hydrocarbons

p-DDOX p-diphenol: dioxygen oxidoreductase

PHDB Petroleum hydrocarbon degrading bacteria

POPs Persistent organic pollutants

PSB Phosphate solubilizing bacteria

RCA Reinforced clostridial agar

SAFs Spacecraft assembly facilities

SGB Second generation biofuel

SGBs Second generation biofuels

SIDA Sabourand dextrose agar

SNA Starch nitrate agar

SPDC Shell petroleum development company

SRB Sulfate reducing bacteria

SCC Salmonella shigella count

TB Translational bioinformatics

TBI Translational Bioinformatics

TCBS Thiosulphate citrate- bile salts-sucrose agar

TCC Total coliform count

TFC Total fungal count

THBC Total heterotrophic bacteria

THC Total hydrocarbon content

THDB Total hydrocarbon degrading bacteria

THDF Total hydrocarbon degrading fungi

TBI Traumatic brain injury

TOC Total Organic Carbon

TPH Total petroleum hydrocarbon

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TSC Total Staphylococcus count

TVC Total vibrio count

UV Ultra-violet

VP Versatile peroxidase

Acronyms Meaning

ʯ Micro

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