MOLECULAR AND PHYSIOLOGICAL STUDIES ON BACTERIAL ...eprints.covenantuniversity.edu.ng/1155/2/PRELIM...B. Sc. (NAU), M. Sc. (UNILAG), MIPAN. A thesis submitted in partial fulfillment

MOLECULAR AND PHYSIOLOGICAL STUDIES ON

BACTERIAL DEGRADATION OF POLYNUCLEAR AROMATIC

HYDROCARBONS

NWINYI, OBINNA CHUKWUEMEKA (CUGP050150)

B. Sc. (NAU), M. Sc. (UNILAG), MIPAN.

A thesis submitted in partial fulfillment of the requirements for the award of the Degree of Doctor

of Philosophy in Microbiology in the Department of Biological Sciences, School of Natural and

Applied Sciences, College of Science and Technology, to the School of Post-Graduate Studies,

Covenant University, Ota, Nigeria.

July, 2012

ii

DECLARATION

I, NWINYI, Obinna Chukwuemeka, hereby declare that this thesis is a product of my own

unaided research work. It has not been submitted, either wholly or in part, to this or any other

institution for the award of any degree, diploma, or certificate. All sources of scholarly

information that were used in this thesis were duly acknowledged.

…………………………………………….

NWINYI, Obinna Chukwuemeka

iii

CERTIFICATION

We certify that this thesis entitled “Molecular and Physiological Studies on Bacterial

Degradation of Polynuclear Aromatic Hydrocarbons” is an original research work carried out

by NWINYI, Obinna Chukwuemeka (CUGP050150) of the Department of Biological Sciences,

Covenant University, Ota, Nigeria under the supervision of Prof. O.O. Amund and Dr F.W.

Picardal. We have examined and found the research work acceptable for the award of a degree of

Doctor of Philosophy in Microbiology.

……………………………………… …………………………….

Supervisor Date

Prof. O. O. Amund

……………………………………… ……………………………

Co-Supervisor Date

Dr. F.W. Picardal

……………………………………… …………………………….

HOD, Biological Sciences Date

Prof. L.O. Egwari

……………………………………… ……………………………

External Examiner Date

Prof. E.I. Chukwura

……………………………………… ……………………………

Dean, College of Science and Technology Date

Prof. F.K. Hymore

iv

DEDICATION

This work is dedicated to my Lord, my Saviour, Jesus Christ, the Son of the Ever Living,

Almighty God to whom I owe completely my survival to this day and also the grace and

inspiration to complete this work that was very daunting.

v

ACKNOWLEDGEMENTS

I am very thankful to God Almighty and to my saviour Jesus Christ who has never left or

forsaken me at any stage of my development but has always taken care of the totality of my life. I

am grateful to the Chancellor of Covenant University, Dr. David Oyedepo, whose vision to

establish this University provided me the stage to embark on the Ph.D. programme. My immense

thanks also go to my Vice-Chancellor, Prof. Aize I. Obayan, and to the University management

for driving the post-graduate programme of this citadel of learning. My profound gratitude goes

to the Institute of International Education (IIE) and the Fulbright scholarship board for having

found me worthy to be awarded the Junior Fulbright fellowship that enabled my travel to the

United States of America for the experimental part of this research. I am also thankful for the

technical and financial support from the city of Bloomington (Indiana) Parks and Rrecreation,

Department during this research.

I want to sincerely appreciate my kind and laudable research supervisor, Professor O.O.

Amund, for his special interest in my success. God used him as the key that opened linkages to

research laboratories abroad. I thank him for his attention to detail in reading through this thesis.

He was never tired of imparting me with remarkable skills, which he has acquired over a long

period of hard work and unrelenting commitment. I will be committing a terrible mistake, if I

forget to mention the role of Dr. Flynn. W. Picardal, Associate Professor at the School of Public

and Environmental Affairs, Indiana University, United States, who accepted to host me in his

research laboratory, with unrestricted access to all that I needed for my research during my 10

months of intensive research. He was a wonderful host- supervisor to me and I gained a lot from

his impactful research experiences.

You all are not just supervisors but men of deep knowledge with passionate hearts. It is my desire

that the Almighty God will reward your labour of love beyond measure now and even in eternity,

in Jesus’ name (amen).

My deep appreciation goes to the Dean of the School of Post-Graduate Studies, Prof.C.O.

Awonuga, the Dean of College of Science and Technology, Prof. F.K. Hymore, the Deputy-Dean,

vi

School of Natural and Applied Sciences, Prof. L.O. Egwari, and all members of their team for

their indefatigable contributions to the development of Covenant University. I wish to offer my

humble and heart-felt gratitude to my Head of Department, Professor L.O. Egwari, who happens

to be the Deputy-Dean School of Natural and Applied Sciences, for his fatherly attitude, prayers,

guidance and keen interest throughout my research work. May God keep blessing you. I also want

to thank all my past Head of Dpeartment’s for all their encouragement and moral support. I am

fully indebted for the care, support and encouragement from Prof. K.O. Okonjo, Prof M.A.

Mesubi, Prof M.O. Ilori, Prof (Mrs) Oyawoye, Dr. Sunday Adebusoye and all my colleagues and

Faculty of the Department of Biological Sciences, Covenant University.

Good and reliable friends remain as gems to one’s life. Among them are my wonderful

research fellows in the US: Anirban Charkraborty, Thuy An, and Agineszka Furmann. I

appreciate their team spirit and the high level of togetherness that they displayed while we worked

together as office /laboratory mates.

I must not fail to express my deep thanks to friends who ensured that I enjoyed a stress-

free stay during my period of rigorous laboratory work in US. among them are Peter Arenner,

Victor Chijioke, Michael Brown, Kyle Miller, Benjamin Pheasant, Elan Rajamani, Ed Huff and

all the SOCC coffee hour friends; then my friends in Nigeria who have touched my life in special

ways during this period of research: Dr. O.O Ajani, Dr. S.N. Chinedu, Dr. S. Oranusi, Mr J.A.

Adekoya, Mr. O. Oluwagbemi, Mr Onyeka Emebo, Mr O.C. Iroham and Mr Uchenna Efobi.

I am fulfilled having Dr Nnamdi I. Nwinyi, Dr (Mrs) Chinyelu N. Uzoamaka, Nkiru and

Chibuzo as brothers and sisters. You all have been a source of support and encouragement. And

to my wonderful sister- in- law the current Deputy-Vice Chancellor (Academics), University of

Abuja, Dr (Mrs) F.C Nwinyi, Dr Onyekachi Uzoamaka, Arc. and Mrs Muyiwa Odufalu, I say a

big “thank you all” for your encouragement.

My appreciation goes to my wonderful parents, Chief. S.C.O Nwinyi and Mrs R.O Nwinyi and

to my parents in-law, Chief A. Obianinwa and Mrs N. Obianinwa for their parental blessings and

consistent prayer over my life. You are the best parents in the whole world. No amount of money

can pay back you for all your sacrifices towards ensuring good upbringing and worthy character

vii

enshrined in me. Finally, I am sincerely grateful to my beloved wife, Barrister. Chinelo

Elochukwu Nwinyi , for her sweet words of love and care and for her ever willingness to stay by

me during the rigours of this research, even at the expense of her career as a lawyer. She stood

strong and took total charge of the home front while I was away abroad to complete the

experimental aspect of this work. You are indeed a golden pillar in my life. I bless God forever

for giving me a rare gems and jewels of immeasurable value in the persons of my lovely

daughters, Blessing and Miracle. I am grateful for their unconditional love and for their

continuously reminding me to always do them proud.

I will forever love you all.

God bless you all!!!!

viii

CONTENTS

Page

Title i

Declaration ii

Certification iii

Dedication iv

Acknowledgements v

Table of Contents viii

List of Figures xv

List of Tables xix

List of Plates xxi

Glossaries of Terms xxii

Abbreviations xxiii

Physical Symbols xxiv

Abstract xxv

CHAPTER ONE

1.0 INTRODUCTION

1.1 Background of the Study 1

1.2 Statement of Problems 9

1.3 Justification of the Study 14

1.4 Purpose/Objectives of the Study 18

CHAPTER TWO

2.0 LITERATURE REVIEW 20

2.1 Polynuclear Aromatic Hydrocarbons (PAH) and the Biosphere 20

2.2 Structure and Properties of PAHs 21

2.3 Polynuclear Aromatic Hydrocarbons as Soil Contaminants 22

2.4 Sources and Sinks 23

2.5 Sources and Environmental Fate of PAHs 24

2.6 Controls on PAH Assemblages in the Environment 25

2.7 Health and Environmental Concerns 26

2.8 Biodegradation Processes 29

ix

2.9 Remediation Methods and Rate-Limiting Factors 30

2.9.1 Dig and Dump 30

2.9.2 Containment 31

2.9.3 Sustainable Remediation 32

2.9.4 Bioremediation 33

2.10 Factors Affecting Polycyclic Aromatic Hydrocarbon (PAH) Degradation 36

2.10.1 Temperature 37

2.10.2 Effect of pH 38

2.10.3 Effect of Aeration 38

2.10.4 Effect of Salinity 39

2.10.5 Presence of other Organic Matters 39

2.10.6 Bioavailability of PAHs 39

2.10.7 Hydrophobicity and Low Solubility 40

2.10.8 Dissolution of PAH particles 41

2.10.9 Soil Structure and Sorption Mass Transfer 42

2. 10.10 Sorption Rated Mass Transfer 42

2.10.10.1 Diffusion 43

2.10.10.2 Intra Organic Matter Diffusion 43

2.10.10.3 Retarded Intra Particle Diffusion 43

2.10.10.4 Film Diffusion of PAH 44

2.11 Factors Affecting the Rate of Release of Sorbed PAH 44

2.11.1 Ageing Processes 44

2.11.2 Pulverization and Acidification of Soil Aggregates 45

2.11.3 Geosorbent and Sorbent Domains 45

2.12 PAH Degradation- The Role of Biosurfactants 46

2. 12.1 Factors Affecting Biosurfactant Production 48

2.12.1.1 Carbon and Nitrogen Sources 48

2.13. The Role of Plasmids in PAH Degradation 48

2.14 Role of Aromatic Ring Hydroxylating Dioxygenases in PAH Degradation 49

2.15. Cometabolism of PAHs 49

2.15. 1 Cometabolism and the Role of Consortium in PAH Degradation 50

x

2.16 Growth Models and Kinetics in PAH Degradation 52

2.16.1 Substrate-Limited Growth 54

2.17 Isolation of PAH Degraders 54

2.17.1 Isolation by Membrane Filter Method 55

2.18 PAH-Degrading Microorganisms 55

2.18.1 Characteristics of most common PAH Degraders 56

2.18.1.1 Rhodococcus sp. 56

2.18.1.2 Pseudomonas sp. 56

2.19. PAHs catabolic mechanism 60

2.20 Some selected PAHs 67

2.20.1 Naphthalene 67

2.20.2 Anthracene 72

2.20.3 Pyrene 76

2.20.3.1 Pyrene toxicity and its detection 80

2.20.3. 2 Some notable bacterial species involved in pyrene degradation 80

2.20.3.3 Pyrene degradation pathways 81

2.20.3.4. Anaerobic degradation of pyrene and other PAHs 83

2.20.4 Fluoranthene 85

2.20.5 Chrysene 88

2.21 DNA- The Central Dogma of Molecular Biology 89

2.22 Sequencing and their Roles in Phylogenetic Classification 89

2.23 Use of BioEdit version 7.0.9 in Gene Sequence Analysis 95

CHAPTER THREE

3.0 MATERIALS AND METHODS 96

3.1 Chemicals and Reagents 96

3.2 Instrumentation 96

3.3 Preparation of Glassware 97

3.4 Historical Background and Environmental Auditing 97

3.4.1 Microwave extraction of the soil samples using US EPA Method 3546 98

3.4.2 Quantification and Identification of the PAHs using US EPA Method 8270 99

3.5 Soil Sample Collection 100

3.6 Physicochemical Analysis of Soil Samples 101

xi

3.6.1 Determination of Moisture Content 101

3.7 Selection of five PAHs for Enrichment and Isolation of Bacterial Species 102

3.8 Enrichment and Isolation of Bacterial Species 102

3.8.1 Enrichment/Isolation of Bacterial Strains Capable of Utilizing Fluoranthene 103

3.9 Screening for PAH Degraders 104

3.10 Preparation of Stock Solution of Different PAHs 104

3.10.1 Preparation of Naphthalene Stock Solution 104

3.10.2 Preparation of Fluoranthene Stock Solution 105

3.10.3 Preparation of Pyrene Stock Solution 105

3.10.4 Preparation of Anthracene Stock Solution 105

3.10.5 Preparation of Chrysene Stock Solution 106

3.11 Calibration and Calculation of the Retention Time of the Compounds 106

3.11.1 Calculation of Retention Times of the Selected PAHs 106

3.12 Analytical Procedure 109

3.13 Quantification of Cell Numbers During PAH-dependent Growth 110

3.14 Enumeration of Cells 110

3.15 Preservation of the Isolates 111

3.15.1 Preparation of Minimal Salt Salicylic Acid 111

3.15.2 Preparation of 0.2M Minimal Salt Benzoate 111

3.16 Biodegradation Studies 111

3.16.1 Minimal Salts (MS) Medium Preparation 112

3.16.2 Biodegradation and Growth Profile Studies

of Strains OC-1 OC-2, OC-3 and OC-4 on Chrysene, Naphthalene

Pyrene and Fluoranthene 112

3.16.3 Growth Profile Studies and Biodegradation

of Strains CB-1 and CB-2 on Chrysene, Naphthalene Pyrene

and Fluoranthene 114

3.16.4 Growth Profile / Biodegradation of Strains PB-1

and PB-2 on Chrysene, Fluoranthene, Naphthalene and Pyrene 115

3.16.5 Growth profile and Biodegradation Studies of Strains FB-1, FB-2

and FB-3 on Fluoranthene, Anthracene, Naphthalene and Pyrene 116

xii

3.16.5.1 Degradation of Fluoranthene Amended with Blackstrap Molasses

by Strains FB-1, FB-2 and FB-3 118

3.17 Screening for Anaerobic PAH degradation Using Nitrate as a

Reducing Agent in MS Benzoate and Salicylate 119

3.18 Molecular Studies: 119

3.18.1 Construction of the Forward Primer 8FM, Reverse Primers 926R and 1387R 119

3.18.2 Cell Growth for the Isolation of Genomic DNA from Pure Isolates 120

3.18.3 Isolation of the Microbial DNA 120

3.18.4 Isolation of Soil and Sediment DNA 123

3.18.5 Concentration of the genomic DNA 125

3.19 Amplification of 16S rDNA or 16S rRNA 126

3.19.1 Preparation of Primer Solutions for PCR 126

3.19.2 PCR Product Clean Up Using Nucleotide Removal Kit 128

3.19.3 Quantification of the Nucleic Acids Using Spectrophotometry 129

3.19.3.1 Measurement of Absorbance at 260 nm 129

3.19.3.2 Quantification of the Nucleic Acids Using NanoDrop

Spectrophotometry ND-1000 129

3.19.3.3 Agarose Gel Electrophoresis 130

3.20 16 S rRNA Sequencing 131

3.20.1 Editing and Assemblying of Sequences 132

3.21 Long-Term Preservation of the Strains 134

CHAPTER FOUR

4.0 RESULTS 135

4.1 Goegraphic Information System (GIS) Mapping of Pollutant Distribution at

McDoel Switchyard 135

4.2 Physicochemical Analysis of McDoel Switchyard Soil 149

4.3 Isolation of Bacterial Species 150

4.4 Degradation of Naphthalene, Chrysene, Fluoranthene and Pyrene by

Bacterial Strains OC-1, OC-2, OC-3 and OC-4 152

4.4.1 Degradation of Naphthalene 152

4.4.2 Degradation of Fluoranthene 156

4.4.3 Degradation of Pyrene 160

xiii

4.4.4 Degradation of Chrysene 163

4.5 Degradation of Pyrene, Chrysene, Naphthalene by Strains of PB-1 and PB-2 166





4.6 Degradation of Fluoranthene, Naphthalene, Molasses and Fluoranthene, Pyrene

and Anthracene by Strains FB-1, FB-2 and FB-3 178


4.6.2 Degradation of Anthracene 181



4.6.5 Degradation of Fluoranthene Supplemented with Black strap Molasses 190

4.7 Degradation of Chrysene, Pyrene, Naphthalene and Fluoranthene

by Strains CB-1 and CB-2. 193





4.8 Screening for Anaerobic Degradation using Nitrate as Electron Donor

in MS Benzoate and Salicylate Media 205

4.8.1 Anaerobic Degradation in MS Benzoate 205

4.8.2 Anaerobic Degradation in MS Salicylate 208

4.9 Molecular Characterization and Preservation of the Isolated Genomic Sequences 211

4.9.1 Microbial DNA Amplification using Polymerase Chain Reaction (PCR)

and gel electrophoresis 211

4.9.2 Isolation and Quantification of Total Microbial DNA directly

from the Different Soil Samples and their PCR Amplification 215

4.9.3 Analysis of the Sequences using Bioinformatic Tools 220

xiv

CHAPTER FIVE

DISCUSSION 233

Conclusion 256

Recommendations 258

Contributions to knowledge 260

REFERENCES 261

APPENDIX 292

Appendix 1 292

Appendix 2 326

Appendix 3 345

xv

LIST OF FIGURES

Figure Page

1.0 Aromaticity on the Benzene Ring 2

2.0 Sample of Creosote in a Conical Flask 23

2.2 Main Processes and Factors Affecting PAH Degradation 37

2.3 PAH Degradation Intermediate Metabolites 63

2.4 Pathways for the Microbial Degradation of PAHs 64

2.5 Pathways for the Degradation of Catechol 65

2.6 The Tricarboxylic Acid Cycle (TCA) 66

2.7 Structure of Naphthalene 70

2.8 Pathways for the Degradation of Naphthalene 71

2.9 Structure of Anthracene 74

2.10 Pathways for the Degradation of Anthracene 75

2.11 Pathways for the Degradation of Pyrene 84

2.12 Structure of Fluoranthene 86

2.13 Fluoranthene Degradation Pathways 87

2.14

2.15

2.16

Structure of Chrysene

The Central Dogma of Life

The Phylogenetic Tree of Life

88

89

94

3.1

3.2

Annotated Diagram of a PCR Process

Sample of a FASTA file

127

134

4.1 Satellite Imagery of McDoel Switchyard Site Showing Estimated

Concentration of Sub-Surface Lead

138

4.2 Satellite Imagery of McDoel Switchyard Site Showing Estimated

Concentration of Surface Lead

139

4.3 Satellite Imagery of McDoel Switchyard Site Showing Estimated Sub-

Surface Arsenic Concentration

140

4.4 Satellite Imagery of McDoel Switchyard Site Showing Estimated Surface

Arsenic Contamination

141

xvi

4.5

4.6A

4.6B

4.6C

Satellite Imagery of McDoel Switchyard and Estimated PAH Contamination

Analysed soil samples showing estimates of the contaminants at the McDoel

Switchyard


Switchyard


Switchyard

142

143

144

145

4.7a Degradation of Naphthalene by MS-Benzoate Grown Cells, Strains OC-1,

OC-2, OC-3, and OC-4

154

4.7b Naphthalene–dependent Growth and Cell Numbers of Strains OC-1, OC-2,

OC-3, and OC-4 after 14-day incubation

155

4.8a Degradation of Fluoranthene by MS-benzoate Grown Cells, Strains OC-1, OC-

2, OC-3, and OC-4

158

4.8b Fluoranthene–dependent Growth and Cell numbers of Strains OC-1, OC-2, OC-

3, and OC-4 after 21-day Incubation

159

4.9a Degradation of Pyrene by MS-benzoate Grown Cells ,Strains OC-1, OC-2, OC-

3, and OC-4

161

4.9b Pyrene–dependent Growth and Cell numbers of Strains OC-1, OC-2, OC-3 and

OC-4 after 21-day Incubation

162

4.10a Degradation of Chrysene by MS-benzoate Grown Cells , Strains OC-1, OC-2,

OC-3, and OC-4

164

4.10b Chrysene –dependent Growth and Cell numbers of Strains OC-1, OC-2, OC-3

and OC-4 after 21-day Incubation

165

4.11a Degradation of Naphthalene by MS-benzoate Grown Cells , Strains PB-1 and

PB-2

167

4.11b Naphthalene –dependent Growth and Cell numbers of Strains PB-1 and PB-2

after 14-day Incubation

168

4.12a Degradation of Chrysene by MS-benzoate Grown Cells , Strains PB-1 and PB-2

170

4.12b Chrysene–dependent Growth and Cell numbers of Strains PB-1 and PB-2 after

21-day Incubation

171

4.13a Degradation of Fluoranthene by MS-benzoate Grown Cells, Strains PB-1 and 173

xvii

PB-2

4.13b Fluoranthene–dependent Growth and Cell numbers of Strains PB-1 and PB-2


174

4.14a Degradation of Pyrene by MS-benzoate Grown Cells , Strains PB-1 and PB-2 176

4.14b Pyrene–dependent Growth and Cell numbers of Strains PB-1 and PB-2 after 21-

day Incubation

177

4.15a Degradation of Naphthalene by MS-benzoate Grown Cells ,Strains FB-1 FB-2

and FB-3

179

4.15b Naphthalene–dependent Growth and Cell numbers , Strains FB-1, FB-2 and FB-

3 after 14-day Incubation

180

4.16a Degradation of Anthracene by MS-benzoate Grown Cells , Strains FB-1 FB-2

and FB-3

182

4.16b Anthracene–dependent Growth and Cell numbers of Strains FB-1, FB-2 and

FB-3 after 21-day Incubation

183

4.17a Degradation of Pyrene by MS-benzoate Grown Cells , Strains FB-1 FB-2 and

FB-3

185

4.17b Pyrene–dependent Growth and Cell numbers of Strains FB-1, FB-2 and FB-3


186

4.18a Degradation of Fluoranthene by MS-benzoate Grown Cells , Strains FB-1 FB-2

and FB-3

188

4.18b Fluoranthene–dependent Growth and Cell numbers of Strains FB-1, FB-2 and

FB-3 after 21-day Incubation

189

4.19a Degradation of Fluoranthene Supplemented with Black-strap Molasses by MS-

benzoate Grown Cells, Strains FB-1 FB-2 and FB-3

191

4.19b Fluoranthene and BlackStrap Molasses dependent Growth and Cell numbers of

Strains FB-1, FB-2 and FB-3 after 21-day Incubation

192

4.20a Degradation of Chrysene by MS-benzoate Grown Cells, Strains CB-1 and CB-2 194

4.20b Chrysene–dependent Growth and Cell numbers of Strains CB-1 and CB-2 after

21-day Incubation

195

4.21a Degradation of Fluoranthene by MS-benzoate Grown Cells , Strains CB-1 and

CB-2

197

4.21b Fluoranthene–dependent Growth and Cell numbers of Strains CB-1 and CB-2 198

xviii


4.22a Degradation of Naphthalene by MS-benzoate Grown Cells , Strains CB-1 and

CB-2

200

4.22b Naphthalene–dependent Growth and Cell numbers of Strains CB-1 and CB-2


201

4.23a Degradation of Pyrene by MS-benzoate Grown Cells ,Strains CB-1 and CB-2 203

4.23b Pyrene–dependent Growth and Cell numbers of Strains CB-1 and CB-2 after 21-

day Incubation

204

4.24 Transformation of Nitrate in MS benzoate by Strains OC1, OC2, OC3, OC4,

FB1, FB2, FB3 PB1, PB2, CB1 and CB2.

206

4.25 Transformation of Nitrate in MS Salicylate by Strains OC1, OC2, OC3,

OC4, FB1, FB2, FB3 PB1, PB2, CB1 and CB2.

209

xix

LIST OF TABLES

Table Page

1.0 EU and USEPA Regulated PAH Compounds. 7

2.1 Some Biosurfactant–Producing Bacteria 47

4.1.A Analysis of the Different Pollutants at McDoel Switchyard 146

4.1.B Analysis of the Different Pollutants at McDoel Switchyard 147

4.1.C Analysis of the Different Pollutants at McDoel Switchyard 148

4.2 Physico-chemical Composition of McDoel Soil Samples 149

4.3 Analysis of Nitrate Reduction in 2.5 mM MS Benzoate 207

4.4 Analysis of Nitrate Reduction in 2.5 mM MS - Salicylate 210

4.5

16S rRNA Amplification at Different Dilutions

212

4.6

4.7

Estimation of the total 16S rRNA gene sequence using Nanodrop

Spectrophotometer

Amplification Total Microbial 16S rRNA obtained from the different soil

sample

216

217

4.8.1 GenBank Probe Analysis of 8FM, 926R and1387R Amplified Fragments

of 16S rRNA gene for Strain CB-1

221

4.8.2 GenBank Probe Analysis of 8FM, 926R and 1387R Amplified Fragments of

16S rRNA gene for strain CB-2

222

4.9.1 GenBank Probe Analysis of 8FM,926R and 1387R Amplified Fragments

of 16S rRNA gene for Strain FB-1

223

4.9.2 GenBank Probe Analysis of 8FM, 926R and 1387R Amplified Fragments


224



225


of 16S rRNA gene for Strain OC-1

226



227

xx



228



229


of 16S rRNA gene for Strain PB-1

230


of 16S rRNA gene for Strain PB-2

231

4.12 Summary of All the Strains of Isolates, their Length, Bacteria Subdivision

and Genbank Accession Number

232

xxi

LIST OF PLATES

Plates Page

3.1 Physical Appearance of the Soil 100

4.1 Fluoranthene Degrading Isolates on Minimal Salt (MS) Agar Sprayed with Fluoranthene:

7 days of Incubation

150

4.2 Strain OC-1 Epifluorescene Microscopy using Acridine Orange Direct Count

(AODC) for Cell Number Enumeration

151

4.3 Flaking of Chrysene 151

4.4 Ethidum Bromide Stained Gel Electrophoresis of Amplified 16SrRNA of Strains

OC4, FB1, FB2 and the marker (1kb DNA Ladder)

213

4.5

Ethidium Bromide Stained Gel Electrophoresis of Amplified 16SrRNA of Strains

OC-1 , OC-2, OC-3 and the marker ( 1kb DNA Ladder)

214

4.6 Ethidium Bromide Stained Gel Electrophoresis of Amplified 16SrRNA of Total

Microbial DNA of Soil Sample 1-3 and the marker (1kb DNA ladder, Primers 8FM

and 1387R)

218

4.7 Ethidium Bromide Stained Gel Electrophoresis of Amplified 16SrRNA of Total

Microbial DNA of Soil Sample 1-3 and the marker (1kb DNA ladder, Primers 8FM

and 926R)

219

xxii

GLOSSARIES OF TERMS

Amplification: a process of multiplying a fragment of DNA by subjecting it to different cycles

of temperature using a thermal cycler in the presence of an enzyme called DNA polymerase.

Anthropogenic: sources of pollution that occur as a result of human activities.

Bioaugmentation: the direct introduction of microorganisms into a contaminated environment to

enhance the cleanup of such an environment.

Biodegradation: the breakdown of a complex chemical by microorganisms, resulting in a minor

loss of functional groups, fragmentation into larger constituents or complete breakdown to carbon

dioxide and minerals.

Bioremediation: the use of biological agents to clean soils and waters polluted by substances

hazardous to human health or the environment.

Teratogenicity: the extent to which a substance causes damage, reflected in the reproductive

organs or abnormality of embryo and offspring.

Biodegradable: undergoing a biological transformation.

Persistent: not undergoing biodegradation in a certain environment.

Recalcitrant: resisting biodegradation in a wide variety of environments.

xxiii

ABBREVIATIONS

ANT

Anthracene

AODC Acridine orange direct counting method

BLAST

BMB

Basic local alignment search tool

Bead mill bioreactor

DHHS Department of Health and Human Services

DNA

DMSO

Deoxyribonucleic acid

Dimethyl sulphoxide

GC-FID Gas chromatography – flame ionization detector

GC-MS Gas chromatography – mass spectrometry

HPLC High pressure liquid chromatography

HMN

HMW

IRT

Heptamethylnonane

High molecular weight

Inhibitor removal technology

LMW

MS

NAPLs

Low molecular weight

Minimal salt

Non-Aqueous Phase Liquids

NRCC

PAH

PCR

National Research Council of Canada

Polycyclic aromatic hydrocarbons/polynuclear aromatic hydrocarbons

Polymerase chain reaction.

PYRQ

PYRdHD

RNA

pyrene-4,5–dione

cis-4,5-dihydroxypyrene

Ribonucleic acid

SDS

SSU

Sodium dodecyl sulphate

Small subunits

SIM Selected ion monitoring

SOM Sorbent organic matter

UDS

USEPA

unscheduled DNA synthesis

United States Environmental Protection Agency

WHO World Health Organization

xxiv

PHYSICAL SYMBOLS

cm centimeter

oC degree celsius

g gram

g/cm2 gram per centimetre square

g/mol gram per mole

μg/ml microgram per millilitre

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mmHg millimetre mercury

mM millimolar

MHz megahertz

ppm parts per million

psi pound per square inch

µL microlitre

µM micromolar

nm nanometer

% percentage

rpm revolutions per minute

ng/m3

nanograms per cubic meter

ng/l nanograms per liter

ppb parts per billion

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ABSTRACT

McDoel Switchyard, an old industrial site in Bloomington, Indiana, US inundated with extensive

levels of organic pollutants, was screened for the presence of Polynuclear aromatic hydrocarbons

(PAHs) degrading bacteria. The incidence of the PAHs and other organic pollutants was

evaluated using the US EPA methods 8270 and 3546. The technique of continual enrichment of

selected PAHs- naphthalene, chrysene, pyrene, fluoranthene and anthracene yielded eleven

unique bacterial isolates tentatively named OC-1, OC-2, OC-3, OC-4, FB-1, FB-2, FB-3, CB-1,

CB-2; PB-1 and PB-2. Following the isolation of pure bacterial strains, each of the bacterial

strains was screened against four different PAHs. Their degradative abilities on the PAHs were

determined using a GC- FID HP 5890 series II gas chromatograph connected to an HP 3396

Series II Integrator. Epifluorescent microscopy was used to measure the cell numbers via a PAH-

dependent growth study. For the molecular characterization of the bacterial strains, genomic

DNA was extracted. The polymerase chain reaction was carried out using 8FM as forward primer,

and as reverse primers 926R and 1387R to amplify the 16S rDNA. The amplified fragments of

16S rDNA were sequenced with an ABI 3730 sequence machine. The results were analyzed using

the following bioinformatic tools: Stuffit Expander 2009, Chromas Lite 2.0, BioEdit,7.0.9, Codon

Aligner and BLAST algorithm. The bacterial strains evolutionary relatedness were performed on

the basis of 16S rDNA gene analysis by comparison of the obtained sequences data with known

sequences in the GenBank.

From the environmental audit carried out at the site of study, two- to three-ring PAHs which were

seven in number were recorded. From the values obtained, the 4-ring PAHs Surface Benzo (a)

anthracene had the highest incidence of about 63,000 μg/kg with minimum and maximum values

between 13.35-63000 μg/kg while the lowest recorded PAH was Sub surface dibenz(a,h)

anthracene with values between 1-1249 μg/kg. Following the screening on the different PAHs, all

the strains showed an ability to utilize a broad spectrum of the different PAHs as carbon and

energy sources. They have also shown an ability to utilize the PAHs under anaerobic conditions.

The biodegradation and PAH-dependent studies showed rapid exponential increase in cell

numbers in some PAHs with about 99% disappearance of some of the PAHs at different volume

biodegradation rates. The 16S rDNA analysis classified the organisms (OC-1, OC-2, OC-3 and

OC-4) as species of uncultured bacterium OC-1, Bacterium OC-2 with about 99% homology to

the type strain of Pseudomonas putida, strain OC-3 as Pseudomonas sp. strain OC3 and OC-4 as

xxvi

Pseudomonas putida strain OC4. Furthermore, strains of FB-1 FB-2 and FB-3 were identified as

Lysinibacillus sp. FB1, Bacterium FB2 with 99% homology to the type strain of Paenibacillus sp.

and Lysinibacillus fusiformis strain FB3 respectively. Strains CB-1 and CB-2 were identified as

Bacterium CB1 with 100% homology to the type strain of an uncultured bacterium and

Stenotrophomonas maltophila strain CB2 respectively. Strains PB-1 and PB-2 were identified as

Pseudomonas plecoglossicida strain PB1 and Pseudomonas sp. strain PB2. The obtained 16S

rDNA gene sequences have been deposited at the GenBank with the accession numbers issued.

MOLECULAR AND PHYSIOLOGICAL STUDIES ON BACTERIAL ...eprints.covenantuniversity.edu.ng/1155/2/PRELIM...B. Sc. (NAU), M. Sc. (UNILAG), MIPAN. A thesis submitted in partial fulfillment

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