molecular and physiological studies on bacterial...
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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
4.5.1 Degradation of Naphthalene 166
4.5.2 Degradation of Chrysene 169
4.5.3 Degradation of Fluoranthene 172
4.5.4 Degradation of Pyrene 175
4.6 Degradation of Fluoranthene, Naphthalene, Molasses and Fluoranthene, Pyrene
and Anthracene by Strains FB-1, FB-2 and FB-3 178
4.6.1 Degradation of Naphthalene 178
4.6.2 Degradation of Anthracene 181
4.6.3 Degradation of Pyrene 184
4.6.4 Degradation of Fluoranthene 187
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
4.7.1 Degradation of Chrysene 193
4.7.2 Degradation of Fluoranthene 196
4.7.3 Degradation of Naphthalene 199
4.7.4 Degradation of Pyrene 202
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
Analysed soil samples showing estimates of the contaminants at the McDoel
Switchyard
Analysed soil samples showing estimates of the contaminants at the McDoel
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
after 21-day Incubation
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
after 21-day Incubation
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
after 21-day Incubation
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
after 14-day Incubation
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
of 16S rRNA gene for Strain FB-2
224
4.9.3 GenBank Probe Analysis of 8FM, 926R and 1387R Amplified Fragments
of 16S rRNA gene for Strain FB-3
225
4.10.1 GenBank Probe Analysis of 8FM, 926R and 1387R Amplified Fragments
of 16S rRNA gene for Strain OC-1
226
4.10.2 GenBank Probe Analysis of 8FM, 926R and 1387R Amplified Fragments
of 16S rRNA gene for Strain OC-2
227
xx
4.10.3 GenBank Probe Analysis of 8FM, 926R and 1387R Amplified Fragments
of 16S rRNA gene for Strain OC-3
228
4.10.4 GenBank Probe Analysis of 8FM, 926R and 1387R Amplified Fragments
of 16S rRNA gene for Strain OC-4
229
4.11.1 GenBank Probe Analysis of 8FM, 926R and 1387R Amplified Fragments
of 16S rRNA gene for Strain PB-1
230
4.11.2 GenBank Probe Analysis of 8FM, 926R and 1387R Amplified Fragments
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.
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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
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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
ml milliliter
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
s seconds
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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
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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.