828S

Biodegradation of Polycyclic Aromatic Hydrocarbons
by Soil Bacteria
By
Fazal ur Rehman
Department of Microbiology
Quaid-i-Azam University
Islamabad
2011
Biodegradation of Polycyclic Aromatic Hydrocarbons

by Soil Bacteria
A thesis submitted in partial fulfillment of the requirements

for the degree of
DOCTOR OF PHILOSOPHY
IN
MICROBIOLOGY
By
Fazal ur Rehman
Department of Microbiology
Quaid-i-Azam University
Islamabad
2011
DECLARATION
The material contained in this thesis is my original work and I have not presented any
part of this thesis/work elsewhere for any other degree.
Fazal ur Rehman
DEDICATED
TO
TO MY LOVING FAMILY MEMBERS
Certificate
The Department of Microbiology, Quaid-i-Azam University, Islamabad,
Pakistan accepts this thesis by Mr. Fazal ur Rehman in its present form as
satisfying the thesis requirements for the Ph.D Degree in Microbiology.
Internal Examiner: ____________________

Dr. Safia Ahmed
External Examiner: _____________________

Dr. Allah Nawaz
External Examiner: _____________________

Dr. Ghazala Kaukab
Chairman:
_____________________
Dr. Safia Ahmed
Dated: May 27, 2011
Table of Contents
Contents
Page
List of Abbreviations
List of Tables
iii
List of Figures
iv
Acknowledgements
ix
Abstract
Chapter 1: Introduction
Chapter 2: Review of Literature
13
Chapter 3: Materials and Methods
32
3.1 CHEMICALS
32
3.2 SELECTION OF PAH TRANSFORMING BACTERIA
32
3.2.1 Culture condition and media
32
3.2.2 Screening of PAHs degrading bacteria
33
3.2.3 Identification of selected bacterial isolates
33
3.2.3.1 Morphological and biochemical characterization
33
3.2.3.2 Molecular characterization based on 16S rRNA
34
gene
3.3 BIODEGRADATION STUDIES OF PAHs
3.3.1 Biotransformation of anthracene and naphthalene using B.
34
34
cereus KWS2 and P. stutzeri 10-1

3.3.2 Resting cell biotransformation of PAHs by Bacillus cereus
35
KWS2 and B. cereus KWS4

3.3.3 Biotransformation of PAHs by Mycobacterium PY146
35
3.3.4 Biodegradation of PAHs in Soil
36
3.3.5 Analytical methods
36
3.3.5.1 Viable Cell Counts
36
3.3.5.2 HPLC Analysis
37
3.3.5.3 Extraction and Analysis of PAHs and their
37
products
3.3.5.4 GC/MS Analysis
37
3.4 MINERALIZATION STUDIES OF PAHs
38
3.4.1 14C-PAH mineralization assay
38
3.4.2 14C-Pyrene and phenanthrene mineralization using
39
different cells densities

3.5 EXPRESSION OF PYRENE DIOXYGENASE GENE (nidAB)
40
FROM MYCOBACTERIUM PY146 into E. coli

3.5.1 Isolation of genomic DNA
40
3.5.2 Amplification of nidAB
40
3.5.3 Cloning of nidAB gene in E. coli
41
3.5.3.1 Cloning of nidAB into pCR 2.1 TOPO vector
41
3.5.3.2 Transformation of E. coli with recombinant pCR
41
2.1 TOPO vector

3.5.3.3 Isolation of recombinant pCR 2.1 TOPO vector
41
from transformed E. coli cells

3.5.3.4 Restriction enzyme analysis
41
3.5.3.5 Addition of restriction sites on the ends of nidAB
42
gene
3.5.3.6 Cloning of nidAB gene in Expression Vector
42
pK223-3
3.5.3.6.1 Dephosphorylation Reaction
43
3.5.3.6.2 Gel Purification of fragments
43
3.5.3.6.3 Ligation of EcoRInidAB HindIII and
43
pkk223-3 fragment
3.5.3.6.4 Transformation of E. coli with recombinant
44
pkk223-3
3.5.3.6.5 Isolation of a recombinant pkk223-3
plasmid containing EcoRInidAB HindIII
44
3.5.3.6.6 Restriction enzyme analysis of
44
recombinant pkk223-3 vector

3.5.3.6.7 Sequencing of the recombinant pk223-3
45
plasmid contained EcoRInidAB HindIII

3.5.3.6.8 Direct Colony PCR to verify fragment
45
insert size
3.5.4 Biotransformation of PAHs by E. coli with cloned nidAB
45
into pkk223-3
3.5.5 Invitro RNA synthesis of nidAB transcripts
46
3.5.5.1 Linearizing the nidAB DNA template
46
3.5.5.2 Synthesis of large quantities of RNA
46
3.5.5.3 Isolation of RNA transcript of nidAB
47
3.5.6 Quantification of DNA and RNA copy number of nidAB

3.5.6.1 Isolation of DNA and RNA from Mycobacterium
47
47
PY146
51
Chapter 4: Results
51
51
51
4.1.1.2 Molecular characterization based on 16S rRNA gene
52
4.2 BIODEGRADATION STUDIES OF PAHS

59
59

60
KWS2 and KWS4

67
4.2.4 Biodegradation of PAHs in soil
73
75
4.3.1
14
C-PAH mineralization by Bacillus cereus KWS2 and
75
Mycobacterium PY146
14
4.3.2
C-Pyrene and phenanthrene mineralization using
75

80
FROM MYCOBACTERIUM PY146

4.4.1 Cloning of nidAB gene
80
4.4.2 Biotransformation of PAHs by E. coli with cloned nidAB
81
into pkk223-3
4.4.3.1 Quantification of RNA transcripts of nidAB from E.
81
81
coli
4.4.4 Comparison of nidAB DNA and RNA copy number using
81
real-time PCR with PAH mineralization in Mycobacterium

PY146
Chapter 5: Discussion
88
Conclusions
101
Future Prospects
102
References
103
Appendices I-V
132
List of Abbreviations
BaA
Benzo(a)anthracene
BaP
Benzo(a)pyrene
BSM
Basal salt medium
BSTFA
N,O-Bis(trimethylsilyl)triflouroacetamide
DGGE
Denaturing gradient gel electrophoresis
DNAPLs
Dense nonaqueous phase liquids
ER
Estrogen receptors
GC/MS
Gas chromatography/mass spectrometry
GEM
Genetically engineered microorganisms
HMW
High molecular weight
HPLC
High performance liquid chromatography
IPTG
Isopropyl--D-thiogalactopyranoside
LB
Luria-Bertani
LNAPLs
Light nonaqueous phase liquids
LP
Lignin peroxidase
MnP
Manganese peroxidase
MR-VP
Methyl red-Voges Proskauer
NADPH
Nicotinamide adenine dinucleotide phosphate
NDO
Naphthalene 1,2-dioxygenase
PCBs
Polychlorinated biphenyls
PCR
Polymerase chain reaction
PAHs
Polycyclic aromatic hydrocarbons
PHE
Phenanthrene
pht
Phthalate degrading operon
PNR
Mineral salt medium
PNRG
PNR with 5 mM Glucose
POPs
Persistent organic pollutants
ppm
Parts per million
QC-PCR
Quantitative competitive polymerase chain reaction
rpm
Rounds per minute
RT-QPCR
Real time quantitative polymerase chain reaction

i
SPM
Suspended particulate matter
TBE buffer
Tris/Borate/EDTA buffer
TCA
Tricarboxylic acid
TPH
Total petroleum hydrocarbons
TSAP
Thermosensitive alkaline phosphatase
TSP
Total suspended particulates
US EPA
United States Environmental Protection Agency
WHO
World Health Organization
ii
List of Tables
Table No.
1.1
4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
4.9
4.10
Caption
Structures, Formulas, MW and solubilities of some
priority polycyclic aromatic hydrocarbons identified
by the US EPA
Growth of bacterial isolates on PNR agar plates
containing anthracene
Growth of bacterial isolates on PNR agar plates
containing Pyrene
Morphological characteristics of bacterial isolates
Biochemical characterization of selected bacterial
isolates
Sugar fermentation tests of the bacterial isolate
Identification of the bacterial isolates on the basis of
16S rRNA
GC mass spectral data of metabolites from
naphthalene,
phenanthrene
and
anthracene
degradation by B. cereus KWS2
naphthalene and phenanthrene degradation by B.
cereus KWS4
phenanthrene, anthracene and pyrene degradation by
resting cells of Mycobacterium PY146
phenanthrene, anthracene and pyrene degradation by
growing cells of Mycobacterium PY146
Page No.
3
53
54
55
56
57
58
65
66
71
72
iii
List of Figures
Figure No.
1.1
1.2
3.1
3.2
4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
4.9
4.10
4.11
4.12
4.13
4.14
4.15
Caption
Mechanisms of action of aromatic dioxygenases. A)
Aromatic ring hydroxylating dioxygenase; B) extadiol
ring cleavage dioxygenase; and C) intradiol ring
cleavage dioxygenase.
Proposed phenanthrene degradation pathway in M.
vanbaalenii PYR-1
Map of plasmid vector pCR 2.1-TOPO used for
nidAB cloning
Map of vector pKK223-3 used for nidAB expression
Percentage reduction of anthracene concentration (10
ppm) in PNRG medium at different pH values by
Bacillus cereus KWS2 in shake flask experiment.
Growth (CFU/mL) of Bacillus cereus KWS2 in
PNRG medium with 10 ppm anthracene at different
pH values.
Percentage reduction of anthracene concentration (10
ppm) in PNRG medium at different temperature by
Bacillus cereus KWS2 in shake flask experiment.
Growth (CFU/mL) of Bacillus cereus KWS2 in
PNRG medium with 10 ppm anthracene at different
temperature.
Percentage reduction of naphthalene concentration by
Pseudomonas stutzeri 10-1
Percentage reduction of anthracene concentration by
Pseudomonas stutzeri 10-1.
Growth of different Mycobacterium strains on 10 ppm
pyrene in BSM medium.
Effect of different temperature on the growth of
Mycobacterium PY146
Effect of different pH on the growth of
Mycobacterium PY146
Biodegradation of PAHs (phenanthrene, anthracene
and pyrene) in soil by Mycobacterium PY146 without
addition of yeast extract.
Biodegradation of PAHs (phenanthrene, anthracene
and pyrene) in soil with addition of yeast extract by
Mycobacterium PY146.
14
C-phenanthrene mineralization by Mycobacterium
PY146
14
C-pyrene mineralization by Mycobacterium PY146
14
C-benzo(a)pyrene mineralization by Mycobacterium
PY146.
Mineralization of 14C-pyrene by cells of
Mycobacterium PY146 along different incubation
time
Page No.
10
11
49
50
62
62
63
63
64
64
69
70
71
74
74
77
77
78
78
iv
4.16
Mineralization of pyrene with different cell densities

79
by Mycobacterium PY146
4.17
Mineralization of 14C-phenanthrene with different cell
79
densities by Mycobacterium PY146.
4.18
Ethidium bromide stained agarose gel showing the
83
PCR product of nidAB gene from Mycobacterium
PY146. Lane L: 1KB DNA ladder; Lanes 1: nidAB.
The arrow indicates the location of the nidAB
product.
4.19
Ethidium bromide stained 1% agarose gel showing the
83
location of the plasmid (top arrow) and nidAB gene
(bottom arrow) after digestion with EcoR1. Lane L:
1KB DNA ladder; Lanes 1, 2 and 5: have both the
plasmid and nidAB.
4.20
Ethidium bromide stained 1 % agarose gel showing
84
the plasmid and nidAB after digestion with EcoR1 and
HindIII. Lane L: 1KB DNA ladder; Lanes 1 and 2
have only plasmid; Lanes 3-8: have both the plasmid
and nidAB.
84
4.21
Ethidium bromide stained 1 % agarose gel showing
the presence of cloned nidAB gene in transformed E.
coli. Lane L: 1KB DNA ladder; Lanes 1 to 6: all have
the cloned nidAB.
85
4.22
Copies of nidAB RNA transcripts in E. coli clones
grown in LB containing ampicillin with and without
IPTG.
4.23
Standard curves of DNA and RNA/ml of bacteria
86
from different cell densities from 0.1 to 0.5 O.D.
4.24
RNA to DNA ratio at different cell densities from 0.1
86
to 0.5 O.D
4.25
Comparison of mineralization of phenanthrene from
87
different cell densities and the nidAB RNA/DNA ratio
in Mycobacterium PYR146.
4.26
Effect of added un-labled pyrene on the mineralization
87
14
of C- labled pyrene at different cell densities of
Mycobacterium PYR146 and the nidAB RNA/DNA
ratio.
5.1
Proposed pathway for the degradation of naphthalene
99
and phenanthrene by B. cereus. Compounds in
brackets are from other reported studies.
5.2
Metabolites detected from the biotransformation of
100
phenanthrene,
anthracene
and
pyrene
by
Mycobacterium PY146
Figures in Appendix
Ia
Phylogenetic tree of bacterial isolate
132
Ib
Percent identity of bacterial isolates
133
Ic
nidAB gene sequence cloned from Mycobacterium
134-135
PY146 and containing the EcoRI and HindIII
restriction enzyme adaptor sequences
II a
II b
II c
II d
II e
II f.
II g
II h
II i
II j
II k
III a
Mass spectra obtained by GC/MS of 2-Naphthalnol

produced by Bacillus cereus KWS2 grown with
naphthalene (A) and from NIST mass spectral
library (B).
Mass spectra obtained by GC/MS of benzeneacetic
acid produced by Bacillus cereus KWS2 grown
with naphthalene (A) and from NIST mass spectral
library (B).
Mass
spectra
obtained
by
GC/MS
of
Benzenecarboxylic acid produced by Bacillus
cereus KWS2 grown with naphthalene (A) and
from NIST mass spectral library (B).
Mass spectra obtained by GC/MS of Bezaldehyde
library (B).
Mass spectra obtained by GC/MS of 9phenanthrenol produced by Bacillus cereus KWS2
grown in phenanthrene (A) and from NIST mass
spectral library (B).
Mass spectra obtained by GC/MS of 9, 10-dihydro9, 10-dihydroxyphenanthrene produced by Bacillus
cereus KWS2 grown in phenanthrene (A) and from
NIST mass spectral library (B).
acid produced by Bacillus cereus KWS2 grown in
phenanthrene (A) and from NIST mass spectral
library (B).
Mass spectra obtained by GC/MS of 4-hydroxy
benzeneacetic acid produced by Bacillus cereus
KWS2 grown in phenanthrene (A) and from NIST
mass spectral library (B).
Mass
spectra
obtained
by
GC/MS
of
benzenecarboxylic acid produced by Bacillus
cereus KWS2 grown in anthracene (A) and from
anthracene (A) and from NIST mass spectral library
(B).
KWS2 grown in anthracene (A) and from NIST
Mass spectra obtained by GC/MS of 2-naphthalenol
library (B).
136
137
138
139
140
141
142
143
144
145
146
147
vi
III b
III c
III d
III f
III g
III h
III i
IV a
IV b
IV c
IV d
IV e

acid produced by Bacillus cereus KWS4 grown
with naphthalene (A) and from NIST mass spectral
library (B).
Mass spectra obtained by GC/MS of Bezaldehyde
library (B).
Mass spectra obtained by GC/MS of 9phenanthrenol produced by Bacillus cereus KWS4
Mass spectra obtained by GC/MS of 9,10-dihyro9,10-dihydroxyphenanthrene produced by Bacillus
cereus KWS4 grown in phenanthrene (A) and from
library (B).
KWS4 grown in phenanthrene (A) and from NIST
benzoic acid produced by Bacillus cereus KWS4
Mass spectra obtained by GC/MS of 1, 2benzenecarboxylic acid produced by resting cells of
Mycobacterium PY146 with phenanthrene (A) and
Mass spectra obtained by GC/MS of 1,2benzenecarboxylic acid produced by resting cells of
Mycobacterium PY146 with anthracene (A) and
Mycobacterium PY146 with anthracene (A) and
Mycobacterium PY146 with pyrene (A) and from
Mass spectra obtained by GC/MS of 1hydroxypyrene produced by resting cells of
Mycobacterium PY146 with pyrene (A) and from
148
149
150
151
152
153
154
155
156
157
158
159
vii
Va
Vb
Vc
Vd
Ve
Vf
Vg
Vh
Vi
Mass spectra obtained by GC/MS of 1,2benzenedicarboxylic

acid
produced
by
Mycobacterium PY146 grown in phenanthrene (A)
and from NIST mass spectral library (B).
acid produced by Mycobacterium PY146 grown in
library (B).
Mass spectra obtained by GC/MS of 9,10anthracenedione produced by Mycobacterium
PY146 grown in anthracene (A) and from NIST
Mass
spectra
obtained
by
GC/MS
of
benzenecarboxylic
acid
produced
by
Mycobacterium PY146 grown in anthracene (A)
anthracene (A) and from NIST mass spectral library
(B).
acid
produced
by
Mycobacterium PY146 grown in anthracene (A)
Mass
spectra
obtained
by
GC/MS
of
benzenecarboxylic
acid
produced
by
Mycobacterium PY146 grown in pyrene (A) and
pyrene (A) and from NIST mass spectral library
(B).
acid
produced
by
Mycobacterium PY146 grown in pyrene (A) and
160
161
162
163
164
165
166
167
168
viii
Acknowledgments
I am grateful to Almighty Allah, the most merciful and beneficent; His blessings enabled me to achieve my
goals. Tremulous veneration is for His Holy Prophet Hazrat Muhammad (PBUHS) who is forever torch of
guidance and knowledge for humanity.
I have great reverence & admiration for my research supervisor Dr. Safia Ahmed. I am highly indebted for
her patience, moral support, sincere criticism and valuable guidance throughout the study.
I am also grateful to Prof. Dr. Abdul Hameed Dean Faculty of Biological Sciences, Quaid-i-Azam
University Islamabad, for providing research facilities at the Department.
Sincere thanks to Prof. Dr. Gary S. Sayler, Director Center for Environmental Biotechnology, University of
Tennessee, USA for providing me an opportunity to work under his kind supervision and valuable guidance.
Thanks to all the CEB fellows for their support and help specially Dr. Fu, Dr. Alice, Dr. Jennifer, Jim and
Jack .
Special thanks are to Dr. Fariha Hasan, Dr. Aamer Ali Shah and Dr. Naeem Ali for suggestions & guidance
that helped me towards the completion of this work.
I highly appreciated the efforts, guidance, and help of Dr. M. Ayub, Dr. Noor Wazir, Masroor Hussain,
Dr.Khalid Mahmood, Khalid Mahsood, Dr. Tatheer, Murad and Zia Ullah for their cooperation and
guidance.
A great depth of loving thanks to my lab fellows Lubna Tahir, Sadia, Naima, Nida, Munzer and Ali for
their help, care and enjoyable company throughout my study, which made me relax during the whole period. I
sincerely acknowledge the friendly attitude & assistance of technical staff of the lab and the office staff of
the department throughout my research work.
I also want to acknowledge Higher Education Commission, Pakistan who provided me the financial support
through indigenous scholarship for my studies and an opportunity for six months training abroad under
IRSIP program.
A non-payable debt to my family members, their wish motivated me in striving for higher education; they
prayed for me, shared the burden and made sure that I sailed through smoothly. Last but not the least; my
endless thanks are to my parents whose motivation is sprinkling added in my way.
Fazal ur Rehman
ix
Abstract
Biodegradation of organic pollutants such as polycyclic aromatic hydrocarbons using
soil microorganisms where they use these compounds as carbon and energy source, is
the safe, cheap and environment friendly way.
In the present study bacterial samples were isolated from crude oil contaminated soil.
A total of 52 isolates were initially screened, fifteen of these isolates were checked for
growth on PNR medium containing up to 1200 and 800 ppm, anthracene and pyrene,
respectively. Five bacterial isolates were selected on the basis of their best on PAHs
growth and identified as Bacillus cereus KWS2, Bacillus cereus KWS4, Bacillus
licheniformis DW3, Bacillus licheniformis Sol-10, and Pseudomonas stutzeri 10-1.
Biodegradation studies of anthracene were carried out by Bacillus cereus KWS2 at
different pH, temperature and incubation time. The optimum pH and temperature
were 7 and 30C respectively, for degradation and growth where 45 % of reduction in
anthracene concentration was observed after seven days of incubation. Growth was
maximum after 2 days of incubation (12 x 107 CFU/mL). Pseudomonas stutzeri 10-1
was also checked for anthracene and naphthalene degradation capability. 33.81 %
reduction in naphthalene concentration was observed after one week, while 33.69 and
45.35 % reduction after two and three weeks time, respectively. In case of anthracene,
87.37 % reduction was observed after first week of incubation whereas 92.98 and
95.56 % was observed after two and three weeks, respectively.
Resting cell biotransformation studies of naphthalene, phenanthrene and anthracene
were carried out by Bacillus cereus KWS2 and KWS4, analyzed by GC/MS. From
naphthalene biotransformation four metabolites, 2-naphthol, benzeneacetic acid,
benzoic acid and benzaldehyde were detected and identified. From phenanthrene
biotransformation,
9-phenanthrenol,
9,10-Dihydro,9,10-dihydroxyphenanthrene,
bezeneacetic acid, 4-hydroxy-benzeneacetic acid and 2-hydroxy benzoic acid were

detected and identified. Benzenecarboxylic acid, benzeneacetic acid and 4-hydroxybenzeneacetic acid were detected and identified from anthracene biotransformation by
B. cereus KWS2. The detection of both mono and dihydroxylated compounds
x
suggested that B. cereus strains KWS2 & KWS4 harbor both mono and dioxygenase
genes.
Biotransformation potential of a selected bacterial isolate, Mycobacterium PY146 for
PAHs was studied. Resting and growing cell biotransformation of PAHs including
phenanthrene, anthracene and pyrene by Mycobacterium PY146 one metabolite i.e,
1,2-benzenedicarboxylic acid (phthalic acid) from phenanthrene and two metabolites,
1,2-benzenedicarboxylic acid and 9,10-anthracendione from the anthracene
biotransformation, where as two metabolites, 1,2-benzenedicarboxylic acid and 1hydroxypyrene were detected and identified from pyrene biotransformation. While in
growing cell biotransformation of phenanthrene, 1,2-benzenedicarboxylic acid and
benzeneacetic acid, from anthracene 9,10-anthracenedione, benzenecarboxylic acid,
benzeneacetic
acid
and
1,2-benzenedicarboxylic
acid
and
from
pyrene
benzenecarboxylic acid, benzeneacetic acid and 1,2-benzenedicarboxylic acid were

detected and identified.
Biodegradation of PAHs including phenanthrene, anthracene and pyrene were
checked in soil by Mycobacterium PY146 without and with yeast extract. After one
week of incubation the detectable concentration of PAHs without yeast extract,
phenanthrene (98 g), anthracene (60 g) and pyrene (56 g) were decreased to 35,
18 and 27 g, and after 2 weeks of incubation all of the detectable PAHs were 0.14,
0.45 and 0.19 g respectively. There was no significant effect of yeast extract on
degradation except phenanthrene, which decreased from 98 to 27 g after one week.
Mineralization studies of different 14C-PAHs (naphthalene, phenanthrene, pyrene and
benzo(a)pyrene) were carried out by Bacillus cereus KWS2 and Mycobacterium
PY146. In case of naphthalene and phenanthrene mineralization by Bacillus cereus
KWS2, no mineralization was observed. Mineralization of phenanthrene, pyrene and
benzo(a)pyrene by Mycobacterium PY146 showed that 45.92, 39.89 and 4.82 % of
14
CO2 was produced and detected in the form of Na214CO3.
The mineralization of
14
C pyrene and phenanthrene with the different cell densities
from OD600 0.1 to 0.5 of Mycobacterium PY146 cultures showed that the maximum
mineralization of pyrene was observed in the OD600 0.2 culture, while in the case of
xi
phenanthrene there is increase in mineralization between the OD600 0.1 to 0.2 culture.
There was subsequent decrease in both pyrene and phenanthrene mineralization as the
cell density increases.
The expression of pyrene dioxygenase gene (nidAB) from Mycobacterium PY146
was checked in E. coli by cloning the nidAB in pKK223-3 expression vector and then
transformed the E. coli. The biotransformation of naphthalene, phenanthrene,
anthracene and pyrene was checked using transformed E. coli cells containing nidAB.
Neither any hydroxy nor any derivatized metabolite were detected of any PAH after
derivatization
with
N,O-Bis(trimethylsilyl)trifluoroacetamide
with
%Trimethylechlorosilane (BSTFA).
Quantification of nidAB RNA transcripts in E. coli was done by RT-QPCR which
revealed an ~ 2- fold increase in RNA transcripts in one sample grown in the presence
of IPTG. RNA to DNA ratio of nidAB calculated by RT-QPCR was increased
between OD600 0.1 to 0.4 and then declined at and OD600 of 0.5. Relation of
phenanthrene and pyrene mineralization to RNA/DNA ratio with the OD600 from 0.1
to 0.5 showed the positive relationship between phenanthrene mineralization and the
RNA/ DNA ratio while in case of pyrene only the OD600 of 0.3 to 0.5 corresponds to
the ratio.
From the present study, it is concluded that soil is the rich source of microbes having
the degradative capability of pollutants like PAHs. Some microbes have the ability to
degrade and mineralize the PAHs efficiently while others can only transform but cant
mineralize. The expression and the RNA transcript of pyrene dioxygenase (nidAB)
copy number and RNA/DNA ratio correspond to the mineralization of PAHs.
xii
Chapter1Introduction
Polycyclic Aromatic Hydrocarbons (PAHs)

Extensive urbanization and rapid industrialization has resulted in the accumulation of
a large number of xenobiotic chemicals into the natural environment. The use of
numerous aromatic compounds in explosives, dyestuffs, pharmaceuticals and
pesticides, and the growth of industrialization have contributed very much in the
environmental pollution and have attracted considerable attention continuously. Many
aromatic hydrocarbons, polycyclic aromatic hydrocarbons, polychlorinated biphenyls,
nitroaromatic compounds, dioxins and their derivatives are highly mutagenic, toxic
and/or carcinogenic to naturally occurring microflora as well as to higher organisms
including humans (Singh and Jain, 2003).
Polycyclic aromatic hydrocarbons (PAHs) are organic substances composed of carbon
and hydrogen atoms grouped into at least two condensed aromatic ring structures.
These compounds have low water solubility, as the number of benzene ring and
molecular weight increases the solubility and degradability decreases (Table 1.1). In
optimum laboratory conditions, half life values of approximately less than three days
for two ring naphthalene have been seen in the soil and water mixture in aerobic
conditions. Half life values for high molecular weight PAHs varies from 30 to 300
days (Al-Turki, 2009).
Sources and Occurrence of PAHs in the environment
Polycyclic aromatic hydrocarbons are produced naturally and by human activities,
therefore they are found commonly everywhere in the environment (Mueller et al.,
1996). Fossil fuels like crude oil and coal are the natural sources, while the partial
burning of the organic matters like vegetation, wood, coal and mineral oils are best
studied examples of PAHs production to the environment by humans (Freeman and
Catell, 1990; Lim et al., 1999). Automobiles are the major contributors to produce
PAHs. The quantity of these contaminants is dependent on the types of reactions and
the fuels used. The concentration of PAHs emission is higher in case of diesel
combustion as compared to petrol or their blended mixture with methanol (Stenberg et
al., 1983). The US EPA include 16 PAHs in their pollutant list, out of these 10 PAHs
are of high molecular weight. These are benzo(a)pyrene, benzo(a)anthracene,
benzo(b)fluoranthene,
benzo(k)fluoranthene,
benzo(g,h,i)perylene,
1
dibenz(a,h)anthracene, chrysene, fluoranthene, pyrene, and indeno(1,2,3-cd)pyrene

(Yan et al., 2004).
Crude oil spills and the industrial wastes generated during coal gasification and
liquefactions for the production of coke are the point sources of PAHs origin (Husian,
2008). Coal tar and creosotes (contain about 85 percent of PAHs), produced during
the coke production have the large amount of PAHs. Burnt food and cigarette smokes
are also the cause of PAHs generation in smaller volumes (Bamforth and Singleton,
2005).
The existence of contaminants like PAHs in soil and sediments pose considerable
hazards to that soil due to the toxicity, mutagenicity and, in some cases,
carcinogenicity associated with the PAHs (Cerniglia and Heitkamp, 1989; Patnaik,
1992). Chemical pollutants present in different parts of the environment slowly
change into more or less harmful products due to natural processes. The process of
degradation depends on various factors particular to the environment of pollutants and
susceptibility of these compounds to degradation (Dabrowska et al., 2005).
Table 1.1: Structures, Formulas, MW and solubilities of some priority polycyclic aromatic
hydrocarbons
identified
by
the
US
EPA
(Keith
and
Telliard,
1979).
(http://chemfinder.cambridgesoft.com/)
PAH
Structure
Formula
MW
Aqueous
Solubility(mg/L
H2O)
Naphthalene
C10H8
128.1732
31.7
Phenanthrene
C14H10
178.233
1.18
Anthracene
C14H10
178.233
0. 043
Pyrene
C16H10
202.255
0.013
Benzo(a)
C18H12
228.2928
0. 014
C20H12
252.3148
0. 0038
C22H12
276.3368
0. 00026
Anthracene
Benzo(a) pyrene
Benzo(g,h,i)
perylene
Environmental Hazards of PAHs

Many PAHs and their oxidative metabolites are highly toxic, mutagenic and
carcinogenic to microorganisms as well as to higher organisms including humans
(Samanta et al., 2002). PAHs have the tendency to accumulate in the organisms at the
end of the aquatic food chain. PAHs and their oxidative metabolite are reported in
many types of samples from different sources (municipal and industrial waste water,
drinking water and rainwater) (Sabate et al., 2001; Maier et al., 2000; Jamroz et al.,
2003). More angular ring structures of the high molecular weight PAHs results in
increased hydrophobicity (Zander, 1983; Harvey, 1997). In general, HMW PAH
molecules are more hydrophobic, toxic and persist longer in the environment
(Cerniglia, 1992).
PAHs are soluble in lipids and easily absorbed from the gastrointestinal tract of
mammals (Cerniglia, 1984). PAHs are thought to be genotoxic only when they are
activated by mammalian cytochrome P450 monooxygenase enzyme, which oxidizes
the aromatic ring to form reactive intermediates such as epoxide and diol epoxide
(Harvey, 1996). The resultant epoxides demonstrated carcinogenicity (Goldman et al.,
2001). Naphthalene can cause acute poisoning in humans may lead to haemolytic
anaemia and nephrotoxicity. Occupational exposure to naphthalene cause dermal and
some ophthalmological changes and have been observed in workers. Phenanthrene is
known to be a photosensitizer of human skin, a mild allergen and also mutagenic to
bacterial systems under specific conditions (Mastrangela et al., 1996). Little
information is available for other PAHs such as acenaphthene, fluranthene and
flourene with respect to their toxicity in mammals. However, the toxicity of
benzo(b)fluoranthene, benzo(a)anthracene, benzo(a)pyrene, benzo(k)fluranthene,
dibenz(a,h)anthracene and indeno(1,2,3-c,d)pyrene has been studied and there is
sufficient experimental evidence to show that all these are carcinogenic (Liu et al.,
2001; Mastrangela et al., 1996).
Fate of PAHs in Environment
The photo oxidation, adsorption to organic matters and microbial degradation are the
probable routes of PAHs in the environment. Beside this they may be deposit in
adipose tissues (Harvey, 1996).
4
Once PAHs are in the soil, they are subjected to a variety of natural weathering
processes. Photooxidation is one of these processes but is of limited value because
once the source of PAHs spilled in the soil, it usually seeps into the soil away from
sunlight (Cofield et al., 2007). Another process that may take place is dissolution.
This process has low significant because much of PAHs have only minimal solubility
in water. Another mechanism that may determine the fate is the adsorption of PAHs
to soil particles. PAHs have characteristic hydrophobic nature; as a result, these
compounds have a tendency to be adsorbed on soil particles, especially on the organic
fraction of solids (Al-Turki, 2009). This type of binding can make the PAHs
unavailable to biological systems hence there is reduction in toxicity but inhibition in
biodegradation. The reason for this inhibition is that they are partially insoluble in the
medium and are not available for degradation, since the bacteria are well known to
degrade those chemical compounds which are soluble in water (Al-Turki and Dick,
2003). The sequestrations are aging stages in which hydrophobic adsorbed substances
show a declining availability to both remediation and chemical extraction (Brion and
Pelletier, 2005).
The primary compounds of PAHs are converted to different metabolites by the
process of degradation before the process reaches an end (Dabrowska et al., 2005).
There are some reported examples of these intermediate metabolite of PAHs which
stimulate the formation of nucleic acid adducts and result in hindering the proper
regulation of genetic material and cause mutations. After entering the body, these
compounds are oxidized in the liver and transform the no polar PAHs to hydroxylated
ones. The dihydrodiols produced during naphthalene biotransformation by microbes
are more toxic than its parent compound because they are more soluble in water
which increases their availability (Wison et al., 1996).
Photodegradation
Photodegradation is a process which occurs in the presence of light and actively takes
place in environment (air and on water surface and soil), but is inefficient deeply in
soil and sediments.
In aquatic environment, chemical and photo oxidation are important routes for abiotic
degradation of PAHs. High molecular weight PAHs having condensed and complex
5
ring structure are usually oxidized by photolysis easily and more rapidly than low
molecular weight compounds. The angularity of the PAHs also affects the photo
oxidation rate, as the linear PAHs such as anthracene are more photo reactive than
phenanthrene (Kochany and Maguire, 1994). Photo oxidation is thought to be the
initial reaction to oxidize the PAHs partially which facilitate the microbes to degrade
it further. The metabolites produced by photo oxidation are thought to be more
biodegradable (Letho et al., 2000). 9, 10 anthracenedione a photo oxidation product
of anthracene is easily degraded by bacteria (Papadoyannis et al., 2002).
Biodegradation
Polycyclic aromatic hydrocarbons oxidizing microbes are present in the environment
everywhere, which includes soil, sediments and lignin containing wood material (Tam
et al., 2002). During the past decade, novel degradation pathways for polycyclic
aromatic hydrocarbons have been proposed by different isolated microbes from the
environment and characterized for their mineralization ability. Some biochemical
pathways for PAHs biodegradation by bacterial isolates have been investigated and
reported, such as for naphthalene (Resnick et al., 1996), phenanthrene (Kiyohara et
al., 1994) and for anthracene and acenaphthene (Dean-Ross et al., 2001; Pinyakong et
al., 2004).
The new approaches of advancements in molecular biology can be used for the
detection of PAH degrading microbes from different environmental samples
(Watanabe, 2001). Technique of DNA-DNA hybridization has been used to identify
and monitor the important crucial populations recovered from the soil environment
(Sayler et al., 1985; Guo et al., 1997). Laurie and Jones, (2000) detected two different
PAH catabolic genotypes from the contaminated soil with PAHs by using quantitative
competitive PCR (QC-PCR). A microbial consortium from soil having the ability to
efficiently mineralize benzo(a)pyrene was analyzed using denaturing gradient gel
electrophoresis (DGGE) profiling of PCR amplified 16S rDNA fragments (Kanaly et
al., 2002a,b).
In the biodegradation mechanisms molecular oxygen play an important role to start
oxidation of the benzene ring of PAHs by enzymatic action. In the very first step,
dioxygenase catalyzed oxidation of arenes normally occurs in aerobic conditions by
6
bacterial multicomponent enzymes to produce cis dihydrodiols. The resulted

hydroxylated compounds are further cleaved by extradiol or intradiol ring cleaving
dioxygenase enzymes to ortho or meta cleavage pathway, which leads the PAHs
metabolites to further conversion to the intermediate compounds of the TCA cycle
(Cerniglia, 1992; Gibson and Parales, 2000; Figure 1.1). The degradation of a
particular PAH by bacteria from a mixture of compounds is often directed by the
differences in their bioavailability. The bioavailable compound will be degraded first
by bacteria. The microbes isolated from environment such as bacteria normally
metabolize a very limited number of PAHs. Bacteria degrade selectively low
molecular weight PAHs first from the mixture. High molecular weight PAHs having
more than four benzene ring like benzo(a)pyrene metabolisms by bacteria is
comparatively less studied. The oxidation of two and three rings PAHs (like
naphthalene, anthracene and phenanthrene) by dioxygenase enzymes of bacteria is
well studied (Peng et al., 2008). Kim et al., (2004) explained the degradation pathway
of phenanthrene which is initiated by dioxygenase (NidAB) (Figure 1.2).
Microbial Metabolism of High Molecular Weight PAHs
The presence of pollutant chemicals in the environment along with microbes triggers
specific genes to be induced which produce enzymes enhance the biodegradation
process (Chen and Aitken, 1999). To overcome the low solubility of hydrophobic
compounds like PAHs, some microbes produce surfactants having both hydrophilic
and hydrophobic parts to enhance the solubility of PAHs (Georgiou et al., 1992).
These surfactants are classified as anionic, cationic, nonionic and zwitter ionic on the
basis of charge on hydrophilic portion (Kosaric, 2001; Christofi and Ivshina, 2002).
The function of surfactants depending on their structure affects the biodegradation by
different ways. As high molecular weight surfactants normally bind strongly to the
different surfaces where as the reduction of surface tension is achieved by the
surfactants having low molecular weight (Ron and Rosenberg, 2001). The presence of
some PAHs mixture also affects the bacterial metabolism by enhancing or inhibiting
its rate (Juhasz et al., 2004) and some PAHs degradative metabolites even inhibit the
growth of some bacteria (Said et al., 2000). The biodegradation of PAHs and its
metabolism by individual bacterial strain or consortium can also be affected the use of
different substrate and bacterial strains (Sutherland et al., 1995).
7
Microbial metabolism of PAHs occurs through oxidation/reduction reactions in which

transfer of electrons take place to the terminal electron acceptors from the substrates
which are highly reduced. In PAHs metabolism, different mechanisms occur in which
these compounds are completely mineralized, co metabolized or partially
transformed. When the PAHs are completely converted to inorganic molecules like
CO2 and H2O, the process is known as mineralization. Fungi mostly transformed the
PAHs to their hydroxylated aromatic acids. Some microbes use one for their energy
and carbon needs for metabolism and transforming another compound like PAHs to
reduce its size without using as source of carbon and energy, the mechanism known
as co metabolism (Alexander, 1999).
Biodegradation of PAHs and their metabolism by microbes leads to the reduction in
concentration which can be analyzed and quantify by different techniques like High
performance liquid chromatography and Gas chromatography coupled with mass
spectrometry. The presence of different microbial enzymes in the medium like
dioxygenase (Cenci and Caldini, 1997), mono oxygenase (Bezalel et al., 1997) and
laccases by fungi (Srinivasan et al., 1995) also indicate the PAHs metabolism.
Microbes or their genes can serve as biomarkers for the detection and metabolism of
different contaminants like PAHs, like Luciferase (lux) gene of several bacterial
species, Vibrio fischeri and Photobacterium, while the presence of lipase activities in
soil is an indication of oil degradation (King et al., 1990; Margesin et al., 1999).
Biodegradation of organic pollutants and its rate of metabolism can be inhibited or
decreased by several factors including the concentration of the substrate, deficient
electron acceptor like O2 and N (Loehr, 1992). Adsorption to the particles of soil and
the less soluble nature of PAHs also affect the degradation rate because of their non
bioavailability (Mackay, 1997; Karimi- Loftabad et al., 1996). Nutrients, water
content and pH values are some critical factors for polluted soil remediation (Kim et
al., 2005; Rolling et al., 2002). The slow release of nonaqueous phase liquids
(NAPLs) phase PAHs to aqueous phase greatly affect biodegradation rate in
contaminated soil (Arora et al., 2009).
Anaerobic PAH Biodegradation

Polycyclic aromatic hydrocarbons are also present in anaerobic environments like
aquifers and marine sediments (Meckenstock et al., 2000; Ohkouchi et al., 1999). The
presence of PAHs in light NAPLs and dense NAPLs ultimately transfer and sink it to
deep in the soil where O2 is not available and needs anaerobic biodegradation. The
anaerobic degradation of PAHs is not completely known yet, but it is clear that
compounds having additional functional groups or substituted PAHs are more
biodegradable than non-substituted compounds. The additional electron donating
groups on substituted compound facilitate the initial aromatic ring oxidation to
provide O2 by splitting H2O molecule (Wilson and Bouwer, 1997; Hutchins, 1991).
In contrast to the metabolism of single aromatic ring hydrocarbons by anaerobic
biodegradation, less data is available of PAHs. However, anaerobic degradation has
observed mostly in microbes isolated from the sediment of marine environment. Little
is known about the initial activation reactions of these PAHs. This is another area of
intense current research activity (Al-Turki, 2009).
The ability of anaerobic biodegradation of PAHs by microbes in the absence of O2 is
now recognized and the naphthalene biodegradation pathway is proposed (Rockne
and Strand, 1998; Rockne et al., 2000). Now several studies reported that many
classes of PAHs were transformed and degraded in the absence of molecular oxygen
by using other electron acceptors like ferrous irons, nitrate or sulphate and also under
methanogenesis conditions (Widdle and Rabus, 2001). Biodegradation process can be
slower in exposed drier soils on hillocks than in sheltered moist area. Degradation is
slower in cold climates than in more temperate or tropical zones and the degradation
rates generally decrease with depth through soil profile (Al-Turki and Dick, 2003).
Figure 1.1: Mechanisms of action of aromatic dioxygenases. A) Aromatic ring hydroxylating

dioxygenase; B) extadiol ring cleavage dioxygenase; and C) intradiol ring cleavage
dioxygenase.
10
Figure 1.2: Phenanthrene biodegradation pathway in M. vanbaalenii PYR-1 proposed

by Kim et al., 2004.
11
Aim&Objectives
Aim & Objectives

The aim of the project was to study the biodegradation of polycyclic aromatic
hydrocarbons, was achieved by following objectives;
To select PAHs degrading isolates from crude oil contaminated soil and to
characterize them
To study the effect of pH and temperature on the biodegradation of anthracene
To study the growing and resting cell biotransformation of PAHs (naphthalene,
phenanthrene, anthracene and pyrene) by the selected microbes
To determine the mineralization rate of
14
C radiolabled PAHs (naphthalene,
phenanthrene, pyrene and benzo(a)pyrene) by the bacterial isolates

To investigate and identify the genes encoding enzymes for PAH degradation
To isolate, clone and express the pyrene dioxygenase gene of Mycobacterium
PY146 into E.coli
To investigate the relationship between the pyrene and phenanthrene
mineralization and expression of nidA gene of Mycobacterium PY146
12
Chapter2 LiteratureReview
Polycyclic aromatic hydrocarbons contamination
The industrial revolution in late 1700s started the increasing use and dependency of
mankind on fossil fuels to generate electricity, industrial processes and power
transportation. As a result the oil wastes and products can be found in different
compartments of the environment (Vilanova et al., 2001).
The wide spread occurrence of PAHs in the environment is mainly because of their
production and release from the partial combustion of all the organic materials. Once
the compound release to the air by any process, they transported to the far areas and
may deposit on soil particles, plants and in water bodies. The PAHs presence in all the
compartments of the environment creates a major risk to humans as well as to all the
living organisms (Van et al., 1997). The data of total emission of 16 PAHs to the
environment from different countries showed in 2004 that 520 giga grams per year
from biofuel, wild fire and consumer usage (56.7, 17.0 and 6.9 %, respectively) as the
major sources. China, India and United States were the top three with highest PAH
emissions amongst the studied countries (Zhang and Tao, 2009).
On March 24, 1989, the oil tanker Exxon valdez spilled around 11 million gallon of
crude oil in Alaska, the oil spread by tides, winds currents and contaminated
approximately 1200 miles shoreline and affect the sea mammals, birds and other
marine life by killing or injuring (U.S. coast guard, 1993). Peterson, (2001) reported
the affect of oil spill on the habitat and species, they observed the decrease in number
of crabs and sea aster, reduced eelgrass density, increase scavenging birds mortality
and reduction in the fishes population. The MT Tasman Spirit, which was carrying
approximately 62,000 metric tons of crude oil for the Pakistan Oil Refinery, was
ruptured in July 2003 and the spilled oil spread to Clifton beach, Karachi. Blood
profiles from the nearby residents a month after of the spill showed slightly increased
levels of lymphocytes and eosinophils, while the creatinine level in serum and urea
level in blood were in normal ranges. Some skin rash cases in children were observed
(Khurshid et al., 2008). The spill of Tasman Spirit affected badly the marine life and
heavier component of the crude oil such as PAHs were detected in Fishes, Crabs and
Shrimps, Crustaceans, Mollusks and Echinoderms along with the passing time. In
fishes out of 16 PAHs 15 were found and detected (naphthalene, acenaphthylene,
13
acenaphthene,
fluorene,
phenanthrene,
benzo(a)anthracene, chrysene,
anthracene,
fluoranthene,
pyrene,
benzo(b)fluoranthene, benzo(k)fluoranthene and
benzo(a)pyrene benzo(g,h,i)perylene and indeno(1,2,3-c,d)pyrene) (Siddiqi et al.,

2009). The oil spill also affected the marine plants (primary producers) in the coastal
area of Karachi, the most affected group were phytoplankton which is reduced in
abundance as well as in species diversity (Saifullah and Chaghtai, 2005). The acute
health effects were observed in the people exposed to the oil spill area, including
increased frequency of sore throat, sore eyes, headache, breathing difficulties and
nausea/vomiting (Janjua et al., 2006).
World Health Organization (WHO) identified the high concentrations of total
suspended particulates (TSP) in Lahore, Pakistan to be amongst the highest in the
world. The high concentration of TSP is because of the high intensity of polluting
sources (from the many small and large industrial activities, and a diversity of poorly
controlled combustion sources) as well as a lack of effective pollution control. Also,
the arid climate aggravates the high pollution levels, resulting in enormous levels of
suspended particulate matter (SPM). Hi-vol air sampling for yearly mean pollutant
concentrations like metals, ammonium, elemental and organic carbon, various anions
and PAHs associated with particles obtained in Lahore were found greater than one
order of magnitude when compared with those from Birmingham, U.K. These levels
are similar with findings in Karachi, Bombay and Calcutta. Vapours to particle ratios
of PAH in the hotter climate of Lahore were thought to be much higher (Smith et al.,
1996).
Even worse is the situation in cities like Peshawar. Air pollution is one of the major
problems of the city. The increasing population and their transportation means, buses,
cars and trucks produces black soot in very huge amount into the environment which
is the main source of PAHs. Roadside soils are heavily polluted by traffic activities. In
comparison with the PAHs concentration level of roadside soils in other countries
(Jordan, France and Czech Republic), the roadside soil of Peshawar is highly
contaminated with PAHs. Soil samples collected from different roadsides were
analyzed and the individual PAH concentration were ranged from 0.1 to 18.0 mg/kg.
Pyrene and phenanthrene were the most abundant amongst the other PAH
14
compounds. The highest concentration of PAHs was found where the traffic density
was highest; suggesting that the principal source of PAH in the roadside soils is
vehicle emissions (Khan et al., 2008).
The presence and detection of PAHs in food items like fishes, meat, milk, vegetables
and fruits increasing the potential for dietary exposure of human to these compounds.
The sources of compounds are predominantly from environmental pollution and some
food processing. The presence of PAHs on food, mainly like fruits and vegetables due
to contaminated soil, air and water mainly by processes of PAHs adsorption, their
deposition and bioaccumulation (Martens et al., 1997; Chung et al., 2008). In 2003,
Camargo and Toledo, reported the presence of different PAHs in the fruits and
vegetables including; cabbage, lettuce, tomato, grape, pear and apple. Among
different PAHs, benzo(a)anthracene was the most abundant being found in 89% of the
samples. There was no effect on locale in PAHs concentration in fruit while in case of
vegetables, more PAHs detected in roadway sites grown than in the rural area. Shen
and Zhu, (2007) observed the presence of 15 different PAHs in vegetable species and
demonstrated that highest PAHs burden was in leafy vegetables. Bishoni et al., (2006)
reported the presence of PAHs in different vegetables; spinach, cauliflower and potato
in the range of 59.6-194.3 g/kg and fruits; grapes, apple, pineapple, papaya in the
range of 25.5-51.7 g/kg. Ciemniak and Chrachol, (2008) identified 16 PAHs in
different cereal products i.e corn, musli, oats and barley and found the low levels of
PAHs in all the samples. Benzo(a)pyrene was detected in all the tested samples in a
range of 0.0216 g/kg. Salinas et al., (2010) observed different PAHs on the surface
of apples in Mexico and suggested that this deposition was from the contaminated air.
PAHs were also detected in water samples (Tap, river, well and sea water) and fruit
juices in China (Zhao et al., 2009).
DouAbul et al., (1997) reported the presence of 40 different un-substituted and alkyle
substituted PAHs in the fishes from Red sea. The concentration of the total PAHs was
422.1 ng/g of dry weight edible muscles of collected fish samples. The considerable
amounts of different PAHs were detected in the gall bladder and liver of the two
different species of fish (Pointet and Milliet, 2000). Reinik et al., (2007) studied
15
presence of different PAHs including benzo(a)pyrene in meat and meat products and
reported the high concentration of these compounds in smoky and grilled cook meats.
After exposure and absorption of the PAHs by the body, they excreted largely in urine
or feces as hydroxylated metabolites. In addition, because of their lipophilicity, some
portions of PAHs accumulate in the adipose tissue which then may be excreted in
human milk. The breast fed infants are more sensitive to these contaminants than
adults; even the slight concentration may affect the infants health. In a study by a
group in Japan observed and detected the PAHs in commercial and human milk in the
range of 0.232.01 and 0.192.15 mg/kg, respectively (Kishikawa et al., 2003).
Different PAHs in the human breast milk of patients in maternity home after a short
period of delivery and in commercial milk and in their products were detected
(Aguinaga et al., 2007; Hunter et al., 2010).
Toxicity of Poly Aromatic Hydrocarbons
The first recognized chemical carcinogens were the combustion products of coal. In
1775, Percival Pott reported the cancer in human from the coal soot in chimney
sweeps followed by studies in animals in the 1920s and the discovery of carcinogenic
polycyclic aromatic hydrocarbons (PAH), e.g., benzo(a)pyrene (BaP), in the 1930s
(Brown and Thornton, 1957; Searle, 1976). Several PAHs including BaP are reperted
and known to be mutagens, carcinogens, and developmental toxicants in humans.
PAHs, such as BaP suggested to be a carcinogen and involved in cancer development
in humans. The activation of estrogen receptors by PAHs are also reported in several
studies which produce estrogenic metabolites. The activation and induction of ER and
metabolites by PAHs could stimulate the cells propagation in estrogen sensitive cells.
Some PAHs (BaP and benzo(a)anthracene) at concentration of 100 nM or higher
could stimulate the proliferation of carcinoma in breast cells of human (Pliskova et
al., 2005).
Some PAHs are transplacental carcinogens in experimental bioassays, producing
tumors in the liver, lung, lymphatic tissues, and nervous system of the offspring
(Bulay and Wittenberg, 1971). The adduct formation of PAHs with DNA is a
biomarker and indication of increased cancer risk and used as measure for genetic
16
damage. The fetus is more susceptible to PAHs associated damages of DNA (10 fold)
than mother and the exposure to PAHs inutero increases the risks. There should be a
preventive measure to minimize the exposure of PAHs to women with pregnancy and
children (Perera et al., 2005). Human macrophages exposure to PAHs affects their
functional properties like adhesion and endocytosis, reduces the surface integrins and
triggers the apoptosis of macrophages (Grevenynghe et al., 2004). Mane et al., (1990)
studied the effects of PAHs on DNA synthesis of human and rat mammary epithelial
cells and observed the reduce activity. Liu et al., (2010) reported the effect of
occupational exposure of PAHs on foundry workers and found the increased amount
of 1-hydroxypyrene in urine sample and DNA strand breakage in the individuals.
The crude oil and their components have phytotoxic effects on plants (red beans and
corn) growth. The aliphatic hydrocarbons have non-significant effects, while the
PAHs such as naphthalene, pyrene and phenanthrene have the phytotoxic effects and
it increases with the increase in benzene rings (Baek et al., 2004). Low molecular
weight hydrocarbons, benzene, toluene, xylene, indene, styrene and naphthalene
inhibited the germination and growth of different plants (Henner et al., 1999). PAHs
and their N-heterocyclic derivatives were studied and found that PAHs derivatives
have more phytotoxic effect than the parent compounds against plant species Sinapis
alba, T. aestivum and P. vulgaris (Paskova et al., 2006).
Control of PAHs pollution by Biodegradation
The PAHs pollution and associated hazards to the environment could be neutralized
with some conventional strategies including isolation, alteration or removal of
pollutants. The process involves, for example excavation of polluted site soil or
incineration of contaminant compounds. In these processes, expensive technologies
are involved and also the transfer of pollutant from one phase to the other. While in
bioremediation, the use of biological organisms to control the pollution by
transforming the hazardous or toxic compounds to less or non hazardous form with
less energy, time and chemical inputs (Ward et al., 2003).
Microbes like fungi and bacteria degrade PAHs partially, oxidize co metabolically or
mineralized completely. Several bacterial species are reported having the ability to
17
utilize PAHs as the only source for energy and carbon. These bacteria degrade and
transform the PAHs compounds into simple molecules which can be used in central
metabolic pathways (Cerniglia, 1992). The hydrophobic nature of PAHs tends it to
bind with the soil organic matter and non aqueous phases in the environment which
results in the unavailability of the compounds to microbial degradation.
Microorganisms especially bacteria have evolved several different mechanisms to
adapt to these hydrophobic compounds in the polluted area. Some microbes produce
biosurfactant to enhance the availability of less bioavailable compounds, while others
show the chemotaxis to reach the PAHs which improve the biodegradation. The
coexistence of fungi and bacteria ubiquitously in the soil environment and the mutual
catabolic activities of fugal and bacterial species may lead to enhance the degradation
process of PAHs in the environment (Peng et al., 2008).
Fungal biodegradation
A wide variety of fungal species have been isolated from different ecological sources
and shown to metabolize the several recalcitrant organic compounds including PAHs
from two to six benzene rings. Many filamentous fungi have been reported to colonize
the solid substrate and tolerate high concentration of toxic compounds (Cerniglia et
al., 1985; Robinovich et al., 2004). Fungi metabolize the PAHs mainly by the two
ways which are mediated by ligninolytic (white rot fungi) and non ligninolytic fungi
(Hammel et al., 1992).
Fungi also detoxify PAHs and other pollutants by the formation of bound residues by
the process known as humification rendering them unavailable to human and other
organisms (Cerniglia, 1997). Humification is the process by which fungi transform
the organic compounds and incorporate it to the humic substances to reduce their
availability and toxicity (Bollag, 1992). Humic substances play a role as binding
ligand for stable linkage with pollutants and to detoxify the environment. The
humification process is initiated by fungal enzymes, phenol oxidases and peroxidases
(Bollag and Myres, 1992). The detoxification of PAHs by the aid of biological
enzymes and its coupling to humic substances also facilitate the other soil microflora
for further transformation of the compounds (Gianfreda and Rao, 2004).
18
The initial reaction of non ligninolytic fungi in the PAHs metabolism is to oxidize the
benzene ring of the compound to produce arene oxides by monoxygenase enzyme,
cytochrome P450 (Sutherland et al., 1995). The metabolism of PAHs by mammals
also occurred in similar way. Dioxygenase enzymes of bacteria oxidize the aromatic
ring of PAHs to produce cis dihydrodiols, while the monoxygenase enzymes of fungi
incorporate single atom of oxygen to the aromatic ring and subsequently produce
trans dihydrodiols (Jerina, 1983).
Ligninolytic enzymes (laccases and peroxidases) which oxidize the lignin found in
different organic matters especially in wood are produced by the white rot fungi
(Mester and Tien, 2000). The oxidation of wood and other organic material occurs
through reactions based on non specific radicals by these extracellular enzymes (Kirk
and Farrell, 1987). Peroxidase enzymes can be classified to manganese peroxidase
(MnP) and lignin peroxidase (LP) on the basis of their reducing substrate, the LP and
MnP are able to oxidize different PAHs (Mester and Tien, 2000). Phenol oxidase
enyme, Laccases, are also able of oxidizing PAHs (Bamforth and Singleton, 2005).
Chulalaksananukul et al., (2006) isolated a filamentous fungus Fusarium sp. E033
from plant leaves exposed to traffic emissions which have the ability to degrade and
tolerate the high concentration of BaP. A strain of Absidia fusca was isolated from a
pesticide contaminated soil which can remove the fluoranthene and anthracene very
efficiently from the medium approximately 89 and 90 % after 24 hours. Another
strain of A. fusca from uncontaminated soil source also removed the fluoranthene and
anthracene (Villemain et al., 2006). Silva et al., (2009) studied the ability of different
fungal isolates from the rich humic substance soil in microaerobic and very low
oxygen condition for the degradation of PAHs. Naphthalene and phenanthrene were
degraded efficiently by Aspergillus sp, Fusarium oxysporum and Trichocladium
canadense while T. canadense, Aspergillus sp, Verticillium sp and Achremonium sp.
were efficient in degrading chrysene, perylene and naphthol(2,3-a)pyrene.
A white rot fungus, Polyporus sp. S133 isolated from soil contaminated with
petroleum, degrade phenanthrene and BaP efficiently in soil. Polyporu sp. S133
transform the phenanthrene and 9,10-phenanthrenequinone and 2,2-diphenic acid
were detected from the medium, while one metabolite, benzo(a)pyrene-6,12-quinone
19
was also detected from BaP degradation (Hadibarata and
Tachibana, 2009;
Hadibarata, 2009).
Chupungars et al., (2009) reported the fungal isolates Agrocybe sp. CU-43 and
Xylaria sp. CU-1 having the degradation activity of different PAHs. The fungus
Agrocybe sp. CU-43, degraded fluorene, phenanthrene, anthracene, fluoranthene and
pyrene at higher rate than Xylaria sp. CU-1. Agrocybe sp. CU-43 degraded different
concentration of fluorene efficiently upto 750 ppm. Two metabolites 9-fluorenol and
9-fluorenone were detected in fluorene degradation medium, the fungus was able to
degrade 9-fluorenone as well. Siripong et al., (2009) used five previously isolated
fungus from two ecologically different sites to degrade anthracene and 2,4-PCB.
More than 90% degradation was observed by all the isolates.
A white rot fungus, Pleurotus ostreatus has the ability to mineralize catechol,
phenanthrene, pyrene, BaP, anthracene and fluorene at slower rate but did not
mineralize fluoranthene (Bezalel et al., 1996). Bishnoi, et al., (2008) reported the
PAHs degradation capability of Phanerochaete chrysosporium isolated from the
contaminated soil of petroleum refinery which degraded acenaphthalene, anthracene,
phenanthrene, fluoranthene and pyrene in both sterile and unsterile soil.
Two fungal isolates Irpex lacteus and Pleurotus ostreatus were used for remediation
studies of two different soils contaminated with PAHs. Fungal isolates actively
degraded PAHs in both soil samples, but there was little or no effect of surfactants
addition on the overall PAHs degradation (Leonardi et al., 2007). Canas et al., (2007)
reported the effect of natural and synthetic mediators on transformation of different
PAHs by fungal laccase of P. cinnabarinus ss3. Laccase degrade anthracene
efficiently, whereas the natural mediators significantly increased the pyrene and BaP
removal.
Bacterial degradation of PAHs
A number of phenanthrene degrading bacterial isolates from different environments
are reported (Phale et al., 2007). Numerous pyrene metabolizing microorganisms are
also isolated and identified which include gram positive actinomycete, S.
xinjiangenesis that use pyrene as carbon and energy source (Ting et al., 2004). A
20
thermo and halotolerant Bacillus strain DHT which could degrade and utilize pyrene
was also reported by Kumar et al., in 2006. Mueller et al., in 1989, reported the PAHs
can be degraded by different bacterial strain isolated from the contaminated soil in the
form of consortium. This bacterial consortium was able to efficiently metabolize the
high molecular weight PAHs like fluoranthene.
Seo et al., (2007) isolated nineteen different bacterial strains from soil contaminated
with petroleum where different PAHs and their metabolite were detected. Out of
these, five bacterial strains degraded PAHs and also used some organophosphorous as
growth substrate. Daane et al., (2001) isolated different bacteria from the rhizosphere
which were able to degrade different PAHs. The identification and characterization
revealed three groups of bacteria, Pseudomonads, Nocardioforms and Paenibacillus.
The isolation of Paenibacillus which showed no homology with different probes
suggested the novel PAH degrader. They concluded the presence of PAHs degrading
microbes in rhizosphere associated with plants and these can be utilized for
contaminated soil bioremediation.
Fourteen different bacterial isolates were used for the degradation of both
phenanthrene and anthracene. After identification four isolates Thiobacter
subterraneus (EF105547), Escherichia coli (EF105548), Soil bacterium (EF105549)
and Alcaligenes sp. (EF105546) showed efficient degradation activity (Abd-Elsalam
et al., 2009). A microbial consortium YL isolated from sediment samples by
enrichment with pyrene degraded pyrene, fluorene, phenanthrene and fluoranthene
from 8795.9 % after three weeks. Two bacterial strains Bacillus cereus Py5 and
Bacillus megaterium Py6 from the consortium degraded 65.8 and 33.7 % of pyrene,
respectively, in three weeks. Escherichia coli cells transformed with the plasmids of
the enriched consortium degraded 85.7 % of pyrene in three weeks (Lin and Cai,
2008).
Tian et al., (2002) observed that Pseudomonas mendocina strain CGMCC 1.766 grew
well in the presence of different concentration of phenanthrene (20-200 mg/l). They
also detected the 1-hydroxy-2-naphthoic acid as the intermediate metabolite during
bacterial degradation of phenanthrene. Feijoo-Siota, et al., in 2008 studied the
naphthalene biodegradation in sea water by free and immobilized cells of
21
Pseudomonas stutzeri 19SMN4 and reported almost complete degradation of
naphthalene both by free and immobilized cells after six days of incubation at 30C.
Obayori et al., (2008) isolated three different Pseudomonas species from oil
contaminated soil in Nigeria, which utilized a broad range of substrates including
chlorobenzoates, petroleum fractions and different PAHs. All three isolates LP1, LP5
and LP6 degraded pyrene, and removed 67.8, 66.6 and 47.0 %, respectively, from the
medium in 30 days. Pathak et al., (2009) studied the naphthalene degrading potential
of Pseudomonas sp. HOB1, isolated from polluted sediments of Amlakadi canal,
India. The isolated bacterial strain degraded the 2000 ppm concentration of
naphthalene in 24 hours and tolerated high concentration of naphthalene up to 60,000
ppm. A bacterial strain from oil contaminated soil was isolated and identified as
Pseudomonas sp. degraded 74.8 % anthracene in 10 days. The degradation activity of
the isolate was diminished after plasmid curing with acridine orange (Kumar et al.,
2010).
Dean et al., (2001) investigated and comparatively studied the bioavailability of
phenanthrene to two degrading bacteria (Pseudomonas strain R and isolate P5-2) in
the presence of rhamnolipid biosurfactant and with the presence of P. aeruginosa
which produce biosurfactant. There is more mineralization of phenanthrene sorbed
with soil by Pseudomonas strain R than strain P5-2, but in aqueous cultures medium
the rate and extent of phenanthrene mineralization by P5-2 was higher than
Pseudomonas strain R. In sandy loam (which has high phenanthrene sorption
capacity) the addition of rhamnolipid biosurfactant increased the rate phenanthrene
mineralization by Pseudomonas strain R, while the mineralization activity of strain
P5-2 sandy loam minimal and there is no increased mineralization by biosurfactants
addition.
A biosurfactant producing bacteria was isolated from petrochemical contaminated
site, identified as Brevibacillus sp. PDM-3 had the ability to grow on the expense of
phenanthrene, anthracene and fluorene. For phenanthrene degradation after the
optimization of different growth parameters, bacteria showed 93 % degradation in six
days (Reddy et al., 2010). Mallick et al., (2007) reported Staphylococcus sp. strain
PN/Y, isolated from petroleum contaminated soil with the ability to utilize
22
phenanthrene as the only source of carbon and energy. Various metabolites were
identified from the phenanthrene degradation along 2-hydroxy-1-naphthoic acid
suggesting the meta cleavage. The catabolic plasmid, pPHN in strain PN/Y is
identified and confirmed by the loss of degradation activity in the plasmid cured strain
PN/Y and the degradative activity of S. aureus RN4220 transformed with pPHN
plasmid. Burkholderia cocovenenans (BU-3) isolated through enrichment technique
by Wong et al., (2002) showed the high degradation rate of phenanthrene and reduced
more than 95 % concentration of the compound from the medium.
From the data reported on PAHs degrading isolates from soil, it has been observed
that the majority of the degraders belogs to Sphingomonas, which are able to oxidize
and degrade a variety of xenobiotic and natural chemicals, such as diphenylether,
furan, dibenzo-p-dioxin, carbazole, estradiol, polyethylenglycols, chlorinated phenols
and different herbicides, pesticides and biphenyl, naphthalene, fluorene, phenanthrene
and pyrene (Basta et al., 2005). Sphingomonas sp. GY2B isolated from the enriched
consortium from PAH contaminated soil, utilize several PAHs including
phenanthrene, naphthalene, 2-naphthol and salicylic acid as the only source of carbon.
The isolated strain degraded 91 % of phenanthrene in two days, and the metabolites
from phenanthrene degradation identified as 1-hydroxy-2-naphthoic acid, 1-naphthol
and salicylic acid (Tao et al., 2007).
Kasai et al., (2002) studied the bacterial role in the degradation of petroleum and
PAHs in aerobic conditons. They enriched bacteria from sea water by using different
PAHs like 2-methyl naphthalene, phenanthrene, or anthracene as a carbon and energy
source and isolated numbers of genus Cycloclasticus, which could grow on crude oil,
naphthalene, dibenzothiohenes phenanthrene and fluorenes. That bacterial group was
propagated on grains coated with oil and immersed in sea water in beach simulating
tanks. PAHs degradation was observed and as well as the growth of Cycloclasticus
cells on the oil coated grains.
Several Mycobacterium species have been isolated and reported to oxidize and
mineralize the low as well as high molecular weight PAHs. Sepic et al., (1997)
studied the biodegradation of PAHs with different isolates of bacteria derived from an
activated sludge. Mycobacterium sp. prp-I showed an efficient biodegradation
23
potential for three and four ring PAHs. Sepic et al., (1998) studied the biodegradation
potential of two different bacteria; Pasteurella sp. IFA and Mycobacterium using
fluoranthene. After two weeks of incubation at room temperature in aqueous medium
resulted approximately 24% and 46% reduction in fluoranthene concentration was
observed. During that period the bacteria mineralized approximately two third and
four-fifth of biodegraded fluoranthene to CO2 while one third and one fifth of the
original fluoranthene remained as stable metabolic products.
A Mycobacterium sp. isolated from the petrochemical contaminated sediment has the
ability to mineralize pyrene (48 % of pyrene in 3 days) as well as other PAHs like
naphthalene, phenanthrene and fluoranthene. The Mycobacterium sp. also mineralize
the PAHs (2-methylnaphthalen, phenanthrene, pyrene and benzo(a)pyrene ) in
microcosm (Heitkamp et al., 1988a; Heitkamp et al., 1988b; Heitkamp and Cerniglia,
1989). Moody et al., (2001) reported the degradation of phenanthrene and anthracene
by Mycobacterium sp. PYR-1. The bacteria degrade 90 and 92 % of phenanthrene and
anthracene, respectively, in 14 days. Mycobacterium pyrenivorans sp, a pyrene
degrader having a distinctive mycolic acid profile in the cell wall containing epoxymycolates, alpha-mycolates and omega-carboxymycolates and 2-eicosanol (Derz et
al., 2004). The alkaliphilic Mycobacterium which is able to degrade and metabolize
PAHs like pyrene, and utilize it for carbon and energy source (Habe et al., 2004a).
Churchill et al., (1999) reported the mineralization activity of Mycobacterium sp.
strain CH1 isolated from PAHs contaminated sediment of fresh water which
mineralized pyrene, fluoranthene and phenanthrene and used these compounds as
carbon and energy source. Mycobacterium sp. strain A1-PYR degraded pyrene alone
and the degradation activity was increased significantly when fluoranthene or
phenanthrene was added along pyrene (Zhong et al., 2006). Alkaliphilic
Mycobacterium sp. MHP-1 degrades pyrene and Mycobacterium sp. SNP11 utilizes
pyrene, phenanthrene, fluorene and fluoranthene as carbon source (Habe, 2004b).
Enzymes involved in Biodegradation of PAHs
Microbial degradation of PAHs is mediated by oxygenase enzymes, which add one or
two (mono or dioxygenase) hydroxyl groups to the aromatic ring. Several oxygenase
genes were reported from a variety of bacterial isolates in different studies.
24
Monooxgenases are mixed function enzymes which incorporate a single oxygen atom
on to benzene ring of the PAHs (Leveau et al., 1998) and several monoxygenase
enzymes were reported in bacteria for pyrene metabolism which are salicylate
hydroxylase (Katagiri et al., 1996), salicyldehyde dehydrogenase (Schocken and
Gibson) and salicylate 5-hydroxylate (Grund et al., 1992).
Cytochrome P 450, the oxidative enzymes present in bacteria and fungi but produce
slightly different oxidative products symmetrically as in fungi the dihydrodiol
produced will be trans and by bacteria the resultant dihydrodiol will be in cis position.
(Cerniglia et al., 1984). The cis dihydrodiol formation is extremely stereoselective by
dehydrogenases which is NAD dependent (Harayama and Rekik, 1989). The
production of cytochrome P450 enzyme could be inhibited or decreased by some
other liver enzymes which have the ability of PAHs detoxification (Leahy and
Colwell, 1990; Chen et al., 2004).
Microbial dioxygenase enzymes add two atoms of oxygen to the benzene ring of the
PAHs and are classified as ring hydroxylating and ring cleaving enzymes. The PAH
ring hydroxylation enzymes are NADPH dependent and oxidize the PAH ring and
transform it to substituted aromatics by addition of hydroxyl groups. Pyrene and
naphthalene dioxygenase enzymes are the well studied examples (Harayama et al.,
1992).
The extra and intradiol dioxygenase enzymes cleave the aromatic ring of PAHs by
addition of two oxygen and subsequently produced metabolites through meta and
ortho cleavage pathway. Catechols are cleaved by extradiols having single ferrous ion
in active site of a subunit (Harayama and Rekik, 1989). Protocatechuates are normally
cleaved by intradiols and have two sbunits ( and ) and the active site contain
varying ferric ions. The example of intradiols is the salicylate 1,2-dioxygenase from
pseudomonas (Kita et al., 1999; Husain, 2008).
Bacteria having the capability of naphthalene metabolism are found in the
environment enormously which are isolated and from some of these the dioxygenase
enzyme for naphthalene were purified and studied (Peng et al., 2008). Naphthalene
1,2-dioxygenase (NDO), from the Pseudomonas sp. NCBI 9816-4, catalyze the
25
reaction by adding two oxygen to the naphthalene and transform to cis naphthalene
dihydrodiol, using the non heme iron center. NDO consist of two main subunits, and
. subunit of the NDO have six mixed sheets and not involved directly in substrate
specificity or catalysis, while the subunit have 13 helices and 25 strands. Two
distinct domains of subunit, a non heme ferrous iron catalytic and a Rieske domain
having the (2Fe-2S) center. The NDO have a wide range of substrate specificity and
oxidize biphenyl and anthracene to their dihydrodiols (Kauppi et al., 1998; Parales et
al., 2000).
Kasai et al., (2002) showed that phnA gene of Cycloclasticus sp. strain A5 was able to
degrade several the PAHs. They transformed E. coli cells with cloned phnA1A2A3A4
genes had wide substrate specificity and converted several polycyclic including
naphthalene,
phenanthrene,
methylnaphthalene
and
hetetocyclic
aromatic
hydrocarbons dibenzofuran and dibenzothiophene to their subsequent dihydroxylated

compounds. Sphingomonas sp. strain LB126 harbor dioxygenase gene (FlnA1FlnA2), which is responsible for initial attack on fluorene during microbial
degradation. FlnA1-FlnA2 was cloned in E. coli, and transformed cells were able to
oxidize fluorene, Phenathrene as well as some other PAHs (Schuler et al., 2008). The
phdABCD genes cluster encodes phenanthrene dioxygenase in which A encodes for
Large subunit , B for small subunit , C for ferrodoxin and D for ferrodoxin
reductase, from Nocardioides sp. were transferred into Streptomyces lividans. The
transformed cells of S. lividans efficiently coverted the phenanthrene and 1Methoxynaphthalene to their subsequent diol forms (Chun et al., 2001).
Shindo et al., (2001) reported the activity of phenanthrene, toluene biphenyl
dioxygenases genes cloned in E. coil. The transformed E. coil cells with phdABCD
from the Nocardioides sp. KP7 converted the phenanthrene and phenanthridine to
their hydroxylated product. On the other hand E. coli having toluene dioxygenase
(todC1C2BA) from P. putida F1 or biphenyl dioxygenase (bphA1A2A3A4) from P.
pseudoaligenes KF707 were not able to transform their substrates. When E. coli were
transformed with the hybrid of toluene and biphenyl dioxygase substituted subunits
(todC1:bphA2A3A4), the bacteria were able to oxidize dibenzofuran, fluorene and
dibenzothiophene. Pinyakong et al., (2004) reported degradation activity of
26
Sphingomonas sp. A4 that use only acenaphthylene and aceanphthene but not other
PAHs. Genes arhA1 and arhA2 which encode the terminal oxygenase of the ring
hydroxylating dioxygenase were transferred in E. coli cells having the electron
transport protein from Sphingobium sp. P2 having phenanthrene degradation ability.
The recombinant E. coli have specificity towards several PAHs like naphthalene,
phenanthrene, fluoranthene, acenaphthene and acenaphthylene.
In Mycobacterium, pyrene biodegradation is initiated by a pyrene ring-hydroxylating
dioxygenase (nidAB), which encodes and -subunit (Khan, et al. 2001). In
Mycobacterium, nidB is upstream of nidA. This arrangement is unique and
characteristic in the dioxygenase of PAHs degraders. nidAB from the Mycobacterium
vanbaalenii PYR-1 when cloned in E. coli, were functional and transformed the
pyrene to its dihydrodiol form. Since the reductase and ferrodoxin fragments are
required for the electron transfer in the dioxygenase system, dioxygenase might have
borrowed the redutase and ferrodoxin from elsewhere in the cloned E. coli. Brenza et
al., (2003) reported the presence of more than one copies of nidAB gene in other
mycobacterium species which have more than 98 % homology with published
sequence of nidAB of Mycobacterium vanbaalenii PYR-1.
Mycobacterium vanbaalenii PYR-1 possess several different dioxygenases and their
multiple copies besides nidAB. Another dioxygenase gene, NidA3B3 was identified
and isolated from Mycobacterium vanbaalenii PYR-1. The expression of this gene in
E. coli did not show any dioxygenase activity, unless coexpressed with ferrodoxin and
reductase. The recombinant E. coli transformed high molecular weight PAHs such as
pyrene, fluoranthene, benz(a)anthracene, anthracene and phenanthrene to their
dihydrodiols (Kim et al., 2006). Stingley et al., (2004a) identified a phthalate
degrading operon (pht) in Mycobacterium vanbaalenii PYR-1. The putative operon
was 12-19 kb from the nidA. Five different genes in the operon were cloned and
expressed in E. coli, efficiently degraded phthalate to dihydroxyphthalate. For the
identification of additional genes responsible for PAH degradation beside nidAB and
nidA3B3, genomic library of Mycobacterium vanbaalenii PYR-1 was constructed in
fosmid vector. A nidA positive fosmid clone had numerous genes which were
involved in polycyclic aromatic hydrocarbon degradation. The transformed E. coli
27
with fosmid vector converted the phenanthrene to phenanthrene cis-dihydrodiol
(Stingley et al., 2004b).
Sho et al., (2004) reported two distinct gene loci nid and pdo in Mycobacterium sp.
strain S65 which were homologous to nidA. The organizations of catabolic genes
were similar in both loci. In both loci, the nidA homologues have 89 and 99 %
homology with the nidA of Mycobacterium PYR-1. The pyrene catabolic genes of
both loci were expressed only in the presence of phenanthrene and pyrene. Strain S65
degradeg and mineralized the PAHs very efficiently and that activity was thought to
be because of two different set of catabolic genes. A pyrene degrading
Mycobacterium sp. strain CH-2 isolated by Churchill et al., (2008) had two pyrene
dioxygenase genes pdo2AB2 and nidAB and expressed only in the presence of PAHs.
The strain CH-2 mineralized several PAHs including fluoranthene, pyrene and
phenanthrene.
Klankeo et al., (2009) reported that Diaphorobacter sp. and Pseudoxanthomonas sp.
isolated from soil were efficient degraders of PAHs. These isolates had nidA gene on
the plasmid, which was the first report about gram negative bacteria having the nidA.
Beside different dioxygenases, the cytochrome P450 monoxygenase genes
responsible for addition of a single oxygen to an aromatic ring of the PAH in initial
oxidation in the degradation process was reported in Mycobacterium vanbaalenii
PYR-1 by Brezna et al., (2006). Three cytochrome P450 genes CYP151, CYP150 and
CYP51 were identified in strain PYR-1 as well as in other Mycobacterium isolates.
The expression studies in E. coli transformed with CYP151 and 150 showed the
monooxygenation of pyrene and other PAHs.
Bioremediation
Bioremediation is the process by which the hazardous and toxic compounds can be
removed or converted to nontoxic or less toxic forms by the use of biological systems,
such as microbes, which are known for their capabilities of catabolic processes. This
is an efficient and cost effective approach to reduce or completely remove the
problematic organic chemicals from the contaminated environment such as soil and
28
water by microbes. Different type of organic compounds could be removed, but
PAHs, some crude oil products and the derivatives of some organo chlorine can be
removed frequently by bioremediation. The bioremediation process will be successful
if the pollutants are removed or transformed to nonhazardous compounds. The
process lowers the cost and eliminates the need for treating the contaminated soil at
the dumpsite. Under many circumstances, bioremediation is the only economical and
available technology (Dabrowska et al., 2005).
In bioremediation process bacteria, fungi and algae are involved in degradation
through biotransformation and mineralization (into water and carbon dioxide). The
rate of bioremediation depends on the chemical structure, availability and nature of
the pollutant, environmental conditions such as temperature, pH and oxygen. The
abundance, activity and types of microbes also affect the rate of bioremediation
(Singh and Ward, 2004). Many PAHs and other organic compounds are ubiquitous in
the environment and persist longer because of their low solubility and thus are less
bioavailable to microbial degradation. The persistent nature and associated toxic
effects of PAHs result in extensive research on degradation capabilities of
microorganisms led to biodegradation pathways and the knowledge about presence of
PAHs degrading microbes in the environment. These studies increasd the interest to
overcome and remediate the PAHs polluted environment by the use of microbes
(Bamforth and Singleton, 2005).
Soil contaminated with polycyclic aromatic hydrocarbons and phenols can remediate
with biostimulation. Guerin, (1999) reported the removal of PAHs and phenol from
the contaminated soil by the process of stimulation which involves the aeration by soil
mixing and adding low amounts of fertilizers. The microbial population of the soil
increased with biostimulation and the total PAHs and phenol concentration were
significantly decreased in the soil, which confirmed that biostimulation was an
effective process for the remediation of PAHs related contaminated soil. In
bioremediation of PAHs contaminated soil, different factors such as moisture,
surfactants and nutrients addition increased the reduction of PAHs concentration
(Lilia et al., 2008).
29
Biostimulation and bioaugmentation processes were thought to be effective in
remediation. A comparative microcosm study of PAHs contaminated soil revealed
that the concentration of PAHs rapidly decreased in bioaugmented contaminated soil
with fungus Molinia sp. compared to biostimulated soil with ground corn cob (Wu et
al., 2008). Cunningham and Philp, (2000) also reported that bioremediation rate of
diesel contaminated soil was higher in bioaugmentation process with the inoculation
of enriched microbes than the biostimulation strategy by adding fertilizers. Yu et al.,
(2005) reported that bioaugmentation of contaminated sediment slurry with PAHs
degrading bacteria showed no degradation activity, while biostimulation with mineral
salt medium increased the degradation rate.
In bioremediation processes, nutrients are one of the limiting factors, but not in every
case. The addition of nutrients alone to the contaminated soil with coal tar PAHs did
not show any decrease in total PAHs in the lab and also in the field studies, while
addition of biodiesel and nutrients to the contaminated soil increased the removal rate
of PAHs and enhanced bioremediation process by solubilizing unavailable PAHs
(Taylor and Jones, 2001). Sediments contaminated with PAHs amended with nutrients
such as phosphorous and nitrogen, yeast extract, lactate and surfactant tween 80
individually increased the degradation of PAHs, while use of these agents altogether
except yeast extract increased the PAHs degradation rate up to 8 times (Bach et al.,
2005). Gennaro et al., (2008) also observed the addition of tween 80 and inoculum
increased the degradation rate in the bioremediation of PAHs in slurry phase
bioreactor.
A bacterial isolate, Paracoccus sp. strain HPD-2 having PAHs degradation ability
was introduced to the soil contaminated with PAHs in microcosm study. The higher
counts of bacteria, enzymatic activities and microbial biomass as well as reduced
concentration of total PAHs was observed in the bioaugmented microcosm (Teng et
al., 2010). The use of genetically engineered microorganism (GEM) for
bioremediation and to monitor the PAHs contaminants was first introduced by a group
in the University of Tennessee. The P. fluorescence strain HK44 transformed with
naphthalene catabolic plasmid containing lux gene fused in the promoter for catabolic
gene of naphthalene. During degradation of naphthalene lux gene gave the
30
luminescent signal, which could be used for in situ bioremediation monitoring and for
the detection of PAHs like naphthalene in the environment (Cox et al., 2000; Ripp et
al., 2000).
31
Chapter3MaterialsandMethods
3.1 CHEMICALS
Analytical grade pyrene (99 %), anthracene (99 %), naphthalene (99 %) and N,OBis(Trimethylsilyl) trifluroacetamide with 1 % trimethylchlorosilane, phenanthrene
14
(98+ %), indole, ampicillin and
C-labled PAHs (benzo(a)pyrene, pyrene,
naphthalene, phenanthrene and anthracene) were purchased from Sigma-Aldrich and

Fluka (St. Louis, MO, USA). Analytical grade Isopropyl -D-1-thiogalactopyranoside
(IPTG) was purchased from Anatrace (USA). Other chemical and media components
were of high purity and analytical grade from Oxide, Merck and BDH.
3.2 SELECTION OF PAH DEGRADING BACTERIA

3.2.1 Culture condition and media
Nutrient agar and Mineral salt medium was used for initial screening and
transformation experiments. The composition of PNR and PNRG (PNR + 5 mM
glucose) (Khan et al., 2006) is as follows;
Composition of mineral salt medium (PNR) per liter of distilled water
PN (20x) 50 mL used as 50 mL/L
KH2PO4
13.6 % (w/v)
(NH4)2SO4
2.4 % (w/v)
NaOH
2.5 % (w/v)
R salts used as 7ml/L

MgSO4.7H2O
8 % (w/v)
FeSO4.7H2O
0.2 % (w/v)
HCl
0.4 % (w/v)
Agar (1.5 %) was used as solidifying agent.

Basal salt medium (BSM) was used for transformation expreriments by resting
cells. The composition of BSM is as follows;
32
Composition of BSM
NaNo3
4.0 g
KH2PO4
1.5 g
FeCl3
0.005 g
MgSO4
0.2 g
CaCl2
0.01 g
Na2HPO4
0.5 g
dH2O
1000 mL
Media and apparatus were sterilized in an autoclave at 121C and 15 psi for 20
minutes.
3.2.2 Screening of PAHs degrading bacteria

A total of 52 bacterial isolates from the crude oil polluted soil (Malik, 2009) were
grown on nutrient agar plates containing naphthalene (100 ppm) and anthracene (1001200 ppm).
Fifteen strains showing the best growth on nutrient agar plates with anthracene (800
ppm) were screened on PNRG (PNR + 5 mM glucose) and PNR media plates
containing anthracene and pyrene separately, (800-1200 ppm each respectively) as
sole source of carbon and energy. Five isolates were selected on the basis of their best
growth in solid medium with PAHs.

Selected isolates were characterized by colony morphology on nutrient agar, gram
staining and morphological characteristics. Additional biochemical test were
performed according to Bergeys Manual of Determinative Bacteriology (Brenner et
al., 2005) for taxonomic characterization which included gelatine liquefaction, starch
hydrolysis, lipid hydrolysis, casein hydrolysis, H2S production, triple sugar iron,
nitrate reduction, catalase test, lipase test, oxidase test, methyl red, voges proskauer
(MR-VP), citrate utilization and sugar fermentation.
33
Genomic DNA was extracted from strains using the Wizard Genomic DNA kit
(Promega, Madison, WI, USA) using the gram positive bacteria protocol according to
the manufacturer instructions. 16S rRNA genes were amplified using universal
eubacterial primers 8F (5-AGAGTTTGATCMTGGCTCAG) and 1492R (5GGYTACCTTGTTACGACTT-3) (Lane et al., 1985). 25 L PCR reactions
consisted of PuRe Taq Ready-to-go PCR beads (GE Healthcare Buckinghamshire,
UK), 0.5 L of template genomic DNA, 400 nM of each primer. The thermocycler
program consisted of 95C for 10 minutes followed by 40 cycles of: 95C for 30
seconds, 48C for 30 seconds and 72C for 1 minute 30 seconds. The program ended
with a final extension step of 72C for 10 minuntes.
PCR products were column-purified using QIAquick PCR Purification Kit (Qiagen
Valencia, CA, USA). They were sequenced using the universal primer 519F (5CAGCAGCCGCGGTAATAC) on an ABI 3730 DNA Analyzer at the Molecular
Biology Resource Facility at the University of Tennessee, Knoxville, TN, USA.
High quality sequences (~950 bp) imported into the DNASTAR Lasergene v.7
software package, aligned using MegaAlign, and a phylogenetic tree was constructed
using ClustalW (Thompson et al., 2003) program contained within Lasergene.
Pairwise distances between sequences (used for phylogenetic tree construction) were
also exported in tabular format.
3.3 BIODEGRADATION STUDIES OF PAHs

Two selected bacterial isolates (KWS2 and 10-1) were evaluated for their ability to
transform naphthalene and anthracene. Inoculums were prepared in nutrient broth by
growing the cells at 30C overnight and used to inoculate the PNR and PNRG
medium (100 mL) containing PAHs with 10 % v/v. cultures were incubated at 30C at
150 rpm in shaking incubator. Effect of pH and temperature was determined on the
biodegradation experiment by conducting the transformation at different pH (4, 5, 6,
34
7, 8 and 9) and temperature (30, 37, 45 and 50C) in PNRG medium. Samples were
collected from the flasks at time intervals, 0, 24, 48, 72, 96, 120, 144, 168 hrs and
monitored for growth and degradation by viable cell count and disappearance of
PAHs by HPLC analysis respectively.

Resting cells biotransformation of PAHs (naphthalene, phenanthrene and anthracene)
by B. cereus KWS2 and B. cereus KWS4 was checked by growing the cells to 0.5
O.D (600 nm) in 1/4th conc. of LB with 10 ppm of selected PAH in 1000 mL
medium. The cells were harvested by centrifugation at 5000 rpm for 10 minutes and
washed twice with basal salt medium (BSM) and then used as an inoculum for 100
mL of BSM, with 50 ppm of PAHs (each in separate experiment) and incubated at
30C in a shaking incubator for 2 days. The PAHs were extracted from cultures and
analyzed by GC/MS.

Seven different Mycobacterium strains which were previously isolated at the Center
for Environmental Biotechnology, University of Tennessee (Knoxville, TN, USA)
from PAHs contaminated sediment by enrichment culture technique with pyrene as
the sole source of carbon and energy (Debruyn, 2008) were screened for growth in
Basal Salt Medium (BSM) with 0.05 % yeast extract and 10 ppm pyrene.
From these seven isolates Mycobacterium PY146 was chosen for further study. The
optimal growth temperature for this strain was determined by growing it in LB broth
at different temperatures (25, 30, 37C) in a rotary shaker at 150 rpm. The growth was
monitored by measuring the absorbance at 600 nm using spectrophotometer. The
growth of Mycobacterium PY146 was also monitored in LB broth using varying pH
values of 5, 6, 7 and 8. Cultures were incubated at 30C on a rotary shaker at 150 rpm
and the growth was monitored spectrophotometrically at 600 nm.
35
For resting cell biotransformation experiments, the Mycobacterium PY146 was grown
to 0.5 O.D in 1/4th conc. of LB with 10 ppm of selected PAHs in 1000 mL medium.
The cells were harvested by centrifugation and washed twice with BSM and then used
as an inoculum into 100 mL of BSM, with 50 ppm of individual PAHs and incubated
at 30C in a shaking incubator for 2 days. For growing cell biotransformation
experiments, the bacterial strains was grown overnight in 1/4th conc. of LB with 10
ppm of selected PAH in 500 mL flask. The cells were harvested by centrifugation and
washed twice with BSM. The cells were added to 100 mL BSM up to 0.05 O.D (600
nm), with 50 ppm of individual PAHs and incubated at 30C in a shaking incubator
for one week. The PAHs were extracted from these cultures and analyzed by GC/MS.

Biodegradation of phenanthrene, anthracene and pyrene using Mycobacterium PY146
was evaluated in soil. Clean uncontaminated soil (PCB Soil PR3-7) was collected,
dried, sieved and sterilized by autoclaving three times at temperature of 121C and
pressure of 1 atm. Clean soil (8 gram) and White quartz Sand (Sigma) (4 gram) were
mixed and placed in 40 ml EPA vials. Into triplicate vials for each sample, 120 g of
phenanthrene and 72 g of anthracene and pyrene each were added in and mixed
thoroughly. The samples were inoculated with 0.3 O.D (at 600 nm) of Mycobacterium
PY146 cells in six ml of BSM. The experiment was set up with and without 0.05 %
yeast extract. For each experiment, abiotic and biotic controls were prepared. The
abiotic control was same as the test samples, except without any added
Mycobacterium PY146 cells. The biotic control consisted of killed cells of
Mycobacterium PY146 with 1.0 mL of 2 N H2SO4. All vials were incubated at 30C
and at pH 7 in a horizontal shaker in dark. Whole sample contained in the individual
vials were extracted at 0 time, one week and two weeks.
3.3.5 Analytical methods

3.3.5.1 Viable Cell Counts
Viable cell counts were done to monitor bacterial growth by using serial dilutions and
spread plate technique.
36
3.3.5.2 HPLC Analysis
Aliquots (1 mL) were centrifuged at 10000 rpm for 10 minutes and supernatant was
filtered by 0.2 m filter and kept for analysis. Analysis was performed by HPLC
(Waters) with a C-18 reverse phase column (thermo Hypersil 1504.6 mm 5
Hypersil). Anthracene was eluted in isocratic mode from column with acetonitrile and
deionized water (60:40) as eluent at a flow rate of 1.2 mL/min. The concentration of
naphthalene and anthracene was determined at 254 nm by comparison to standard
curve.
3.3.5.3 Extraction and Analysis of PAHs and their products
For extraction of PAHs and their metabolites, 100 mL of the cell suspension were
harvested. After centrifugation at 4C (5000 rpm, 15 min), the supernatant was
collected and extracted three times with equal volumes of ethyl acetate. All extracted
ethyl acetate were pooled together and generated neutral extract. The remaining
supernatant was further acidified to pH 2.5 with 1 M HCl to produce acidic extract in
the same manner. All of the collected ethyl acetate fractions were evaporated
separately at 40C using a rotary evaporator (Rotavapor RE121 Bchi, Switzwrland),
and the residue was dissolved in a 1 mL of acetone. The samples were dried in
vacuum and stored at 20C until used (Kim et al., 2005). Samples were analyzed by
GC/MS.
Phenanthrene, anthracene and pyrene were extracted from soil by adding an equal
volume of ethyl acetate and shaken for an hour on a horizontal shaker. This mixture
was allowed to settle for 10-15 minutes, and then 1 mL of organic phase was collected
from vial and stored at -20C till analysis. Analysis of biodegradation was performed
using GC/MS under SIM mode.
3.3.5.4 GC/MS Analysis
Samples were analyzed by GC/MS (Agilent 6890 Gas Chromatograph with 5973N
Mass Spectrometer with inert source) equipped with a DB5-MS column (30 m x 0.25
mm. i.d., 0.25 m film, (J&W Scientific) according to U.S. EPA Method 8270D.
The GC/MS operating conditions were as follows:
37
Mass range:
35-500 amu
Scan time:
1 sec/scan
Initial temperature:
60C, hold for 4 min
Temperature program:
60-300C at 10C/min
Final temperature:
300C
Injector temperature:
250C
Transfer line temperature:
280C
Injector:
Splitless
Injection volume:
1 L
Carrier gas:
Helium
GC/MS were equipped with DB5-MS column (30 m x 0.25 mm i.d., 0.25 m film).

3.4.1 14C-PAH mineralization assay
Radiolabeled
14
C-PAHs mineralizations were carried out using naphthalene,
phenanthrene and anthracene for Bacillus cereus KWS2 and naphthalene,

phenanthrene, pyrene and benzo(a)pyrene for Mycobacterium PY146. The assay was
performed to determine the production of 14CO2 from 14C-PAHs. Cells were grown in
a shaking growth chamber (150 rpm) in of the LB medium to an O.D 600 of 0.8.
Cells were washed once with BSM and resuspended in BSM broth to an O.D 600 of
0.30. Five mL of the suspension was added to a 40-mL EPA vial. A CO2 trap (4-mL
vial) containing 1.0 mL of 0.5 M NaOH was placed inside the EPA vial. Before the
EPA vial was sealed with a Teflon-lined silicone septum, an appropriate amount of
14
C-PAH dissolved in acetone was added: 33,650 dpm benzo(a)pyrene, 60,550 dpm
pyrene, 78,210 dpm naphthalene, 81,100 dpm phenanthrene and 78,100, dpm
anthracene.
The assays were performed in triplicate with abiotic and biotic controls. Abiotic
controls consisted of BSM media amended with the same radioactive PAH as the test
samples and without any added bacterial cells. Biotic controls consisted of media
containing the radioactive PAH and cells killed with 1.0 mL of 2 N H2SO4. The vials
were incubated at 30C with shaking at 150 rpm for 7 days. The assays were stopped
by injecting 1.0 mL of 2 N H2SO4 through the septa. Then the vials were shaken for 1
38
h to drive 14CO2 into the NaOH traps. The NaOH was mixed in 10 mL of ReadySafe
liquid scintillation cocktail (Beckman Coulter, Fullerton, CA, USA). Mass balances
were achieved by measuring radioactivity from
14
CO2, the liquid media, and the
biomass/residue portion of the mineralization assay. The
14
C-radioactivity was
measured with a TRI-CARB 2900TR liquid scintillation analyzer (PerkinElmer,

Shelton, CT 06484-4794 USA).
3.4.2 14C-Pyrene and 14C-phenanthrene mineralization using different

cells densities
The mineralization of
14
C-pyrene checked with different incubation times starting
with 0.25 O.D of Mycobacterium PY146. The mineralization of
14
C-pyrene and
phenanthrene was monitored using Mycobacterium PY146 cultures ranging in O.D at

600 nm from 0.1 to 0.5. In initial, culture were grown by inoculating the cells in of
the LB medium with 10 ppm of pyrene while shaking (150 rpm) at 30C to an O.D of
0.5 at 600 nm. That culture was transferred to different flasks containing of the LB
medium with 10 ppm of pyrene and phenanthrene separately. The individual flasks
were grown to an O.D of 0.1, 0.2, 0.3, 0.4 and 0.5 at 600 nm. The cells were harvested
with centrifugation and resuspended in the same volume of BSM broth with 0.05 %
yeast extract. Five mL of the suspension was added to a 40-mL EPA vial. A CO2 trap
(4-ml vial) containing 1.0 mL of 0.5 M NaOH was placed inside the EPA vial. Before
the EPA vial was sealed with a Teflon-lined silicone septum, an appropriate amount
of
14
C- 4,5,9,10-pyrene (128188 dpm) and
14
C-phenanthrene (70000 dpm) dissolved
in acetone was added.

The assays were performed in triplicate with abiotic and biotic controls. Abiotic
controls consisted of BSM media amended with the same radioactive PAH as the test
samples and without any added bacterial cells. Biotic controls consisted of cells killed
with 1.0 mL of 2 N H2SO4. All vials were incubated at 30oC with shaking at 150 rpm
for 20 hours. The assays were stopped by injecting 1.0 mL of 2 N H2SO4 through the
septa. Then vials were shaken for 1 h to drive
14
CO2 into the NaOH traps. The
14
C-
radioactivity was measured with a TRI-CARB 2900TR liquid scintillation analyzer.
39

FROM MYCOBACTERIUM PY146 into E. coli
3.5.1 Isolation of genomic DNA
The bacterium was grown in LB broth at 30C for 72 hours. Cells were pelleted by
centrifugation for 10 minutes at 1000 rpm. Genomic DNA was isolated according to
the protocol of FastDNA Spin Kit for soil (MP Biomedicals, Solon, OH, USA). The
isolated Genomic DNA was run on 1 % agarose gel to verify size.
3.5.2 Amplification of nidAB

The pyrene dioxygenase gene (nidAB) was amplified using a forward (5ATGAACGCGGTTGCGGTCGAT-3)
and
reverse
primers
(5-
TCAAGCACGCCCGCCGAATGC-3) and a 25 L PCR reactions consisting of:

Genomic DNA
1 L of template
dNTPs
1 L
Primers (Forward and reverse)
1 L (each)
Buffer A (10X)
2.5 L
Taq polymerase
1 L
dH2O
17.5 mL
The thermocycler program consisted of 94C for 5 minutes followed by 32 cycles of:
94C for 1.0 minute, 60C for 1.0 minute and 72C for 1 minute 30 seconds. After
PCR completion the amplified products were stored at 4C. To check the amplified
fragment size, PCR product and 1kb ladder was run on 1 % agarose gel in 1x TAE
buffer at 90 V.
Composition of 50x TAE buffer:
Tris
242 g/L
NaEDTA.2H2O
18.61 g/L
Glacial acetic acid
57 mL
40
3.5.3 Cloning of nidAB gene in E.coli

3.5.3.1 Cloning of nidAB into pCR 2.1 TOPO vector
A polyA tail was added to both ends of the nidAB gene fragment obtained by PCR
using polyA beads and incubating the mixture at 72C for 30 minutes. The polyA
prepared PCR product (4 L) was then combined with 1 L of salt solution (1.2 M
NaCl, 0.06 M MgCl2) and 1 L of TOPO vector (having ampicillin resistant gene as
selectable marker) (Invitrogen, Carlsbad, CA, USA) (Figure 3.1) and incubated for 30
minutes at room temperature.
3.5.3.2 Transformation of E. coli with recombinant pCR 2.1 TOPO vector
For transformation 6 L of the recombinant nidAB- pCR 2.1 TOPO vector was added
to a vial of competent TOPO10 E. coli cells and kept on ice for 30 minutes. The
reaction vial was heat shocked at 42C for 30 seconds and placed back on ice for 2
minutes. One ml of nutrient rich YT 2x medium (yeast extract + tryptone) was added
to the cells and the cells were incubated at 37C for 1 hour in a shaking water bath.
Aliquots consisting of 50, 100 and 200 L of the transformed cells were spread on LB
agar plates (having ampicillin (100 g/mL) and incubated overnight at 37C. Only the
transformed cells containing the plasmid with ampicillin resistance were able to grow
on the plates.
3.5.3.3 Isolation of recombinant pCR 2.1 TOPO vector from transformed E. coli
cells
The LB broth (0.5 mL) (having ampicillin (100 g/mL)) was inoculated with
transformed E. coli from the agar plates. Samples were centrifuged at 10000 rpm for
10 minutes. The pellet was saved and the plasmid was isolated from the cell pellet
according to the protocol of Promega Wizard miniprep DNA purification System Kit
(Promega, Madison, WI, USA).
3.5.3.4 Restriction enzyme analysis
The isolated plasmid DNA was analyzed by restriction enzyme analysis. One L of
EcoR1, 2 L of Buffer H and 17 L of plasmid DNA were mixed together and
41
incubated for 1 hour at 37C. The reaction products were checked by ruining on a 1 %
agarose gel.
3.5.3.5 Addition of restriction sites on the ends of nidAB gene
New
primer
sequences
were
CTGCAGAATTCGCCCTTATGAACG-3
AAGCTTTCAAGCACGCCCGCC-3
designed,
and
nidABEco
nidABHind
For
5-
Rev
5-
so that after PCR amplification the nidAB
gene would have EcoR1 and HindIII restriction sites on the both ends of the gene.
The PCR reaction consisted of:
Plasmid DNA
1 L
Primer (Forward)
1 L
Primer (Reverse)
1 L
dNTPs
1 L
Buffer A
(10x)
2.5 L
Tag polymerase
1 L
dH2O
17.5 L
The thermocycler program consisted of 94C for 5 minutes followed by 32 cycles of:
94C for 1.0 minute, 61C for 1.0 minute and 72C for 1 minute 30 seconds. The
amplified PCR product was kept at 4C until the PCR tube was removed from
thermocycler. To check the amplified fragment size, the PCR product was run on a 1
% agarose gel. The nidAB gene with the attached restriction sites (EcoR1 and
HindIII) was cloned into TOPO10 vector and transferred to competent E.coli cell. The
cells were grown and the recombinant plasmid was isolated from the cells according
to the protocol of Promega Wizard miniprep DNA purification System Kit. The
ligated restriction sites (EcoR1 and HindIII) at the ends of nidAB gene in this plasmid
were confirmed by sequencing of recombinant TOPO vector with M13 primers.
3.5.3.6 Cloning of nidAB gene in Expression Vector pK223-3
To clone the nidAB gene having EcoR1 and HindIII restriction sites into pkk223-3
expression vector (Amersham) (Figure 3.2), both the recombinant TOPO10 vector
42
and pkk-233-3 expression vector were digested separately with the EcoR1 and
HindIII. The digestion reaction consisted of:
Plasmid DNA
20 L
EcoR1
2 L
HindIII
2 L
Buffer E
5 L
dH2O
21 L
Both the digestion reactions were incubated for 2 hours at 37C, then I L of each
enzyme was added to the reaction and incubated in the same condition for additional
one hour.
3.5.3.6.1 Dephosphorylation Reaction
After digestion with restriction enzymes (EcoR1 and HindIII), plasmid pkk233-3 was
dephosphorylated by adding 1 L of TSAP (Thermosensitive Alkaline Phosphatase)
(Promega USA) and incubated at 37C for 15 minutes. The restriction enzyme and
TSAP were inactivated by incubating at 74C for 15 minutes. Both the digestion
reactions were run on a 1.5 % agarose gel without ethidium bromide and DNA
fragments were excised from the gel.
3.5.3.6.2 Gel Purification of fragments
Bands of both fragments (EconidABHind and pkk233-3) were excised with sharp
blade and DNA was extracted and purified using Gene Clean Spin Kit (Q BioGene)
according to manufacturer's instructions.
3.5.3.6.3 Ligation of EcoRInidAB HindIII and pkk223-3 fragment
After digestion and purification the EconidABHind fragment was ligated with
pkk223-3 using DNA ligase. The reaction mixture was incubated at 16C overnight.
The ligation reaction consisted of:
43
Plasmid vector (pk223-3)
1.5 L
EconidABHind fragment
5.75 L
Ligase buffer (10x)
2 L
Ligase
1 L
dH2O
9.75 L
3.5.3.6.4 Transformation of E. coli with recombinant pkk223-3

A -80C frozen culture of Escherichia coli TOPO10 was used for transformation
using the standard protocol. Cells were thawed on ice and 3 L of ligation reaction
was added to the cells and mixed by tapping. The vial was incubated on ice for 30
minutes. Then it was incubated for 30 seconds at 42C in water bath, and quickly
removed and placed on ice. Then 1 mL of YT medium was added in the vial, and
incubated in shaking water bath at 37C for 1 hour. Aliquots of 50, 100 and 200 L
were plated onto LB agar containing 100 g/mL ampicillin. The plates were inverted
and incubated at 37C overnight. Individual colonies were picked and streaked on
fresh LB agar plates containing 100 g/mL ampicillin and incubated overnight at
37C.
3.5.3.6.5 Isolation of a recombinant pkk223-3 plasmid containing EcoRInidAB
HindIII
A loop-full of each ampicillin resistant culture was taken from the agar plates using a
sterilized loop and mixed in an eppendorf tube with 0.5 mL of LB broth, having 100
g/mL ampicillin. The tubes were centrifuged at 10000 rpm for 10 minutes to pellet
the cells. The plasmid DNA was isolated from the pellet according to the protocol of
Promega Wizard miniprep DNA purification System Kit. The concentrations (ng/L)
of plasmid DNA having the EcoRINidABHindIII fragments were measured by Hoefer
DyNA Quant 200 Fluorometer (GE Healthcare Biosciences, Piscataway, NJ. USA).
3.5.3.6.6 Restriction enzyme analysis of recombinant pkk223-3 vector
The isolated plasmid DNA was analyzed by restriction enzyme analysis. The
restriction enzyme reactions consisted of:
44
Plasmid DNA
15 L
EcoR1
1 L
HindIII
1 L
Buffer H
2 L
dH2O
1 L
The reaction mixture was incubated at 37C for 1 hour. Restriction enzyme products
were separated by gel electrophoresis to verify size on a 1 % agarose gel made with
1x TBE.
3.5.3.6.7 Sequencing of the recombinant pk223-3 plasmid contained EcoRInidAB
HindIII
Three plasmid DNAs (clones 6, 7 and 8) were sequenced using the M13 primers on an
ABI 3730 DNA Analyzer at the Molecular Biology Resource Facility at the
University of Tennessee, Knoxville, TN, USA.
3.5.3.6.8 Direct Colony PCR to verify fragment insert size
Direct colony PCR was done using a slight modification of the procedure from PerezPerez and Hanson 2002, to confirm that the right fragment size was cloned into the
pkk223-3 expression vector.
Briefly, the colony was touched with a disposable
pipette tip and immersed into a PCR tube containing 1 L of each primer (nidAB
Forward and Reverse), 1 L dNTPs, 2.5 L of Buffer A (10x), 1 L of Tag
polymerase and 17.5 L of dH2O. The PCR products and a 1kb ladder were run on 1
% agarose gel in 1x TBE.
3.5.4 Biotransformation of PAHs by E. coli with cloned nidAB into pkk223-3
E. coli having cloned NidAB fragment in plasmid pkk223-3 were grown in LB with
IPTG overnight at 37oC. The cells were washed with BSM twice.
Resting cell
assays using naphthalene, phenanthrene, anthracene and pyrene added individually to

the vials were performed as described in section 3.3.2. The samples were extracted
with an equal volume of Ethyl acetate three times and then analyzed on GC/MS as
described in section 3.3.5.3.
45
In order to detect PAH metabolites 1 mL of the extracted samples were derivatized
with N,O-Bis(trimethylsilyl)trifluoroacetamide with 1 % Trimethylechlorosilane
(BSTFA) (Fluka, Switzerland) by adding 50 L of BSTFA to the samples and
incubated overnight at 50C as recommended by manufacturer. The samples were
analyzed on GC/MS as described above in section 3.3.5.3.

3.5.5.1 Linearizing the nidAB DNA template
The nidAB fragment in the TOPO vector was linearized by digestion with BamH1
restriction enzyme. The digestion reaction consisted of:
Plasmid DNA
40 L
Buffer E
5 L
BamH1
2 L
dH2O
3 L
The digestion reaction was mixed and incubated for 3 hours at 37C, and then another
1 L of the BamH1 enzyme was added and the reaction was incubated again at the
same conditions.
3.5.5.2 Synthesis of large quantities of RNA
A T7 reaction were set up, and the reaction consisted of:
Linear DNA template
8 L
RiboMAXTM Express T7 2x Buffer
10 L
Enzyme Mix, T7 Express
2 L
The reaction was mixed gently and incubated at 37C for 30 minutes. After the
transcription reaction was complete, 2 L of Promega RQ1 RNase-free DNase (cat. #
M6101) was added to the reaction and the reaction was incubated for 1 hour at 37C,
to remove the DNA template. For additional clean-up of the in vitro transcription the
protocol of T7 RiboMAX Express Large Scale RNA Production System (Promega,
Madison, WI, USA) was followed. The RNA concentration was measured on
Nanodrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA).
46
3.5.5.3 Isolation of RNA transcript of nidAB
The TOPO10 E. coli cells (clone numbers 6, 7 & 8) containing the nidAB gene
fragment in the expression vector pkk233-3 which were grown in the LB broth
medium containing 100 g/mL of ampicillin with and without IPTG (0.8 mM) at
37C overnight. The RNA was isolated using FastRNA Pro Soil-Direct Kit
following the protocol.
3.5.6 Quantification of DNA and RNA copy number of nidAB

3.5.6.1 Isolation of DNA and RNA from Mycobacterium PY146
Total DNA and RNA was isolated and purified from Mycobacterium PY146 grown in
LB to different cell concentration ranging from 0.1, 0.2, 0.3, 0.4 and 0.5 O.D at 600
nm. The RNA was isolated using FastRNA Pro soil-Direct Kit, while DNA isolation
was done using FastDNA SPIN Kit for Soil (MP Biomedicals, Solon, OH, USA). A
primer and TaqMan probe set was designed for quantifying dioxygenase genes by
targeting the large () dioxygenase subunit (nidA) and was based on a multiple
sequence alignment of the nidA conserved regions from several PAH-degrading
Mycobacterium.
The
forward
and
reverse
primer
sequences
were
5-
TTCCCGAGTACGAGGGATAC-3, and 5-TCACGTTGATGAACGACAAA-3,

respectively.
The
TaqMan
probe
sequence
was
5d
FAM-
TCCTACCCGTCGCCGGTACA-BHQ-1 3.
The real-time PCR reactions consisted of:
2x Brilliant II SYBR QRT-PCR master mix
12.5 L
Forward primer
0.75 L
Reverse primer
0.75 L
TaqMan probe
0.5 L
dH2O
10 L
DNA or RNA Template
2.5 L
47
The quantitative PCR reactions were performed on an MJ Opticon thermocycler (BioRad, Hercules, CA, USA) using the following protocol: 50C for 2 minutes, 95C for
15 minutes, then 40 cycles of denaturing at 94C for 15 seconds and annealing at
54C for 30 seconds. nidAB from Mycobacterium PY146 cloned into TOPO
(Invitrogen) vector was used to create an eight-point standard curve ranging from 101
to 108 gene copies per reaction.
48
Figure 3.1: Map of plasmid vector pCR 2.1-TOPO used for nidAB cloning
49
Figure 3.2: Map of vector pKK223-3 used for nidAB expression
50
Chapter4Results

A total of 52 bacterial isolates were screened for their growth on PAHs from oil
contaminated sites. Fifteen strains showed the best growth on nutrient agar plates with
anthracene (800 ppm). These strains when tested for their growth in mineral salt
medium (PNR) with anthracene and pyrene as sole source of carbon and energy,
different levels of growth were observed. Majority of the isolates showed growth up
to 400 ppm concentration of the PAHs tested. Strain KWS2 showed rich growth up to
1200 ppm anthracene while other strains showing growth up to 1200 ppm include
KWS4, DW3, Sol-10 and 10-1 (Table 4.1). Growth tolerance in mineral salt medium
with pyrene was up to 800 ppm for the same five strains (Table 4.2). So these five
strains were selected as PAH degrading bacteria for further study.

The five bacterial isolates KWS2, KWS4, DW3, Sol-10 and 10-1 were analyzed
taxonomically. The colony morphology of KWS2 and KWS4 were large, offwhite/creamy in color with irregular margins while DW3 and Sol-10 appeared
opaque. The morphology of 10-1 appeared as small to medium in their size, yellowish
in color with regular margins. All the isolates were gram positive except 10-1 which
was gram negative, and all were rod shape and motile. Spore staining revealed the
presence of spores in KWS2, KWS4, DW3 and Sol-10 (Table 4.3).
Biochemical tests revealed that all the isolates showed gelatin liquification and
catalase activity, and the starch hydrolysis, casein hydrolysis and nitrate reduction
were positive for all the isolates except 10-1. Indole production was negative for all
the isolates, while the methyl red and urease test were negative for all isolates except
KWS2. Simon citrate test was negative for all the isolates except 10-1. All the isolates
showed oxidase activity except Sol-10. Voges proskauer reaction was positive for
KWS2 and KWS4 while the other isolates showed negative reaction (Table 4.4).
Sugar fermentation tests showed that all the isolates were unable to ferment lactose
and fructose. Isolate KWS2 showed acid production from arabinose, sucrose, maltose,
mannose, glucose and inositol, KWS4 produced acid from maltose, mannose and
51
Chapter4Results
glucose, DW3 and Sol-10 were positive for acid production from arabinose, maltose
and mannose, 10-1 produces acid from arabinose, xylose, rhamminose, sucrose,
maltose and mannose fermentation. There was no gas production from any of the
above mentioned sugars by any isolate (Table 4.5).
The five selected bacterial isolates were further identified on the basis of sequence
analysis of 16S rRNA. Based on the phylogenetic tree (Appendix I a) and the data in
tabular format which showed the percent identity (Appendix I b), isolates KWS2 and
KWS4 had more than 99 % (99.4 and 99.3 % respectively) homology in 16S rRNA
sequence to Bacillus cereus DQ 884352. Isolates DW3 and Sol-10 had 99.3 and 99.7
% homology to Bacillus licheniformis EU 373408, while isolate 10-1 had 99.3 %
homology to Pseudomonas stutzeri U 26262 (Table 4.6).
52
Chapter4Results
Table 4.1: Growth of bacterial isolates on PNR agar plates containing anthracene
Concentration of Anthracene (ppm)
Bacterial
Isolates
100
120
150
200
250
300
400
500
600
800
1000 1200
SS13
++
++
++
++
++
++
++
SS18
++
++
++
++
++
KWS3
++
++
++
++
++
++
++
++
++
KWS2
+++
+++
+++
+++
+++
++
++
++
+++ +++ ++
DW3
+++
++
++
++
++
++
++
+++ ++
++
++
EC1
+++
+++
+++
+++
+++
++
++
++
KWS 1S
+++
+++
++
++
++
++
++
++
KWS4
+++
+++
+++
+++
+++
+++
+++
+++ +++ +++ +++
++
KWS 5S
++
++
KWS 6
+++
+++
+++
+++
++
++
RED 18
+++
+++
+++
+++
++
++
Sol-10
+++
+++
+++
+++
+++
+++
+++
+++ +++ ++
++
++
10-1
++
++
+++
+++
++
++
++
++
++
++
KWS 5L
PC 1
++
++
++
++
++
++
++
PA 4
++
++
++
++
++
++
++
Slight Growth (+)
Good Growth (++)
Rich Growth (+++)
NO Growth (-)
53
+++
Chapter4Results
Table 4.2: Growth of bacterial isolates on PNR agar plates containing Pyrene
Bacterial
Concentration of Pyrene (ppm)
Isolates
20
50
75
100
200
300
400
600
700
800
SS13
++
++
++
SS18
++
++
++
++
++
KWS3
++
++
++
++
++
++
++
KWS2
+++
+++
+++
+++
+++
+++
+++
+++ ++
++
DW3
++
++
+++
++
++
++
++
++
++
EC1
+++
++
+++
+++
++
++
++
++
KWS1 S
+++
+++
++
++
++
++
++
KWS4
++
+++
+++
+++
+++
+++
+++
++
++
KWS5 l
++
++
KWS 6
+++
++
+++
++
++
++
RED 18
+++
+++
+++
+++
+++
Sol-10
+++
++
++
+++
++
++
++
++
10-1
++
+++
++
++
++
++
++
++
PA 4
++
++
++
++
++
++
++
KWS1 s
++
++
+++
++
KWS1 l
++
+++
++
++
++
KWS3 s
+++
KWS5 l
++
++
++
++
++
54
Chapter4Results
Table 4.3: Morphological characteristics of bacterial isolates

Strain
Gram
Shape
stain
Spore
Motility
stain
KWS2
Rod
KWS4
Rod
DW3
Rod
Sol-10
Rod
10-1
Rod
55
Chapter4Results
Table 4.4: Biochemical characterization of selected bacterial isolates
Isolates
Starch
Hydrolysis
KWS2
KWS4
DW3
Sol-10
10-1
Casein
hydrolysis
Simon citrate
test
Gelatin
Liquefaction
Catalase test
Indole
Production
Methyl Red
Voges Proskauer
Nitrate
Reduction
Urease
Oxidase
56
Chapter4Results
Table 4.5: Sugar fermentation tests of the bacterial isolates
Sugars Lactose Arabinose Fructose Xylose Rhamminose Sucrose Maltose Mannose Glucose Inositol
A
KWS2
KWS4
DW3
Sol-10
10-1
Strains
A = Acid Production
G = Gas Production
57
Chapter4Results
Table 4.6: Identification of the bacterial isolates on the basis of 16S rRNA
Isolates
%age homology
Accession No. of
sequence giving
homology
Identification
KWS2
99.4 %
DQ 884352
Bacillus cereus
KWS4
99.3 %
DQ 884352
Bacillus cereus
DW3
99.3 %
EU 373408
Bacillus licheniformis
Sol-10
99.7 %
EU 373408
Bacillus licheniformis
10-1
99.3 %
U 26262
Pseudomonas stutzeri
58
Chapter4Results
4.2 BIODEGRADATION STUDIES OF PAHS

4.2.1. Biotransformation of anthracene and naphthalene using B.
Biotransformation studies of anthracene were carried out in growing cells shake flask
experiments in PNRG medium containing 10 ppm anthracene. The experiments were
designed to evaluate the effects of incubation time, pH and temperature on the growth
of Bacillus cereus KWS2 and biotransformation of anthracene.
Biotransformation of anthracene by Bacillus cereus KWS2 at different pH values
revealed that, the highest degradation was observed at pH 7 which was almost 40 %
after four days and 45 % after seven days of incubation at 30C. A 30 % degradation
was observed at pH 6 on day 7 while there was a significant decrease in the
concentration of anthracene at pH values 4, 5, 8 and 9 (Figure 4.1).
The growth study showed that there was progressive increase in growth in terms of
CFU/mL from the initial inoculation. The maximum growth was observed at pH 7
after two days of incubation (12 x107) further there was decline in growth as the
incubation time increased. The second highest growth was observed at pH 6 which
was 10.8 x107 CFU/mL after two days of incubation. There was a decrease in growth
below pH 6 and as well as above pH 7 (Figure 4.2).
The optimum temperature for the degradation of anthracene was observed to be 30C
where 45 % reduction in the anthracene concentration was observed after seven days
of incubation. As the temperature increased the percent degradation decreased. There
was 32, 27 and 20 % degradation at 37, 45 and 50C, respectively, after seven days of
incubation (Figure 4.3).
The effect of temperature on growth of Bacillus cereus KWS2 correlated with the
percentage degradation of anthracene and the highest levels of growth was observed
at 30C which was 12 x107 CFU/mL after two days of incubation. The second highest
growth was observed at 37C which was 7 x107. The growth was decreased
significantly with the increase in temperature (Figure 4.4).
59
Chapter4Results
Bacterial isolate Pseudomonas stutzeri 10-1 was also checked for growing cell
biotransformation of naphthalene and anthracene in PNRG medium separately. In
case of naphthalene, 32.81 % reduction was observed after one week of incubation
while 33.69 and 45.35 % reduction was observed after second and third week
respectively (Figure 4.5). In case of anthracene degradation, 87.37 % reduction was
observed after first week of incubation whereas 92.98 and 95. 56 % reduction was
observed in anthracene concentration after two and three weeks, respectively (Figure
4.6).

Biotransformation experiment of PAHs (naphthalene, phenanthrene and anthracene)
was carried out by Bacillus cereus strains KWS2 and KWS4 in BSM medium
containing 50 ppm of each compound separately at 30C and pH 7.
Resting cell biotransformation of naphthalene by Bacillus cereus KWS2 was
evaluated after two days of incubation. Four metabolites were detected and identified
in GC-MS chromatograms from the ethyl acetate neutral extracts from naphthalene
grown culture after two days of incubation which includes; 2-naphthol {MS
fragments: 144(100), 145(12), 116(48),115(95), 89(14), 72(5), 63(10), 50(4), 39(4)},
benzeneacetic acid {MS fragments: 135.9(34), 92(19), 91(100), 65(14), 63(7), 51(5),
39(6)}, benzenecarboxylic acid {MS fragments: 121.9(96), 105(100), 76.9(81),
51(32), 50(25), 43(30)} and benzaldehyde {MS fragments: 106(100), 105(96), 78(20),
77(93), 74(13), 52(9), 51(42), 50(24), 39(6)} (Table 4.7; Appendix II a-d). All
metabolites were identified by comparing to the mass spectra of standards in NIST
mass spectral library (2005).
Biotransformation of phenanthrene was also evaluated after two days of incubations
by Bacillus cereus strains KWS2. A total of four metabolites were produced by
Bacillus cereus KWS2 which were detected and identified by GC/MS, three from the
neutral extracts of ethyl acetate including; 9-phenanthrenol {MS fragment: 194(100),
166(33), 165(90), 164(15), 163(16), 139(9), 82(11)}, 9,10-Dihydro, 9,10dihydroxyphenanthrene {MS fragment: 212(58), 193.9(34), 181(67), 166(51),
165(100), 153(13), 152(24), 138.9(4), 115(6), 82(9), 76.9(10)} where as bezeneacetic
60
Chapter4Results
acid, and 4-hydroxy-benzeneacetic acid, {MS fragment: 151.9(33), 108(8), 107(100),

77(23), 51(7), 39(4)} were detected in the acidic extract (Table 4.7; Appendix II e-h).
Degradation of anthracene by resting cells of B. cereus KWS2 after two days of
incubation gave three metabolites. These metabolites were detected in acidic extracts
which includes benzenecarboxylic acid, benzeneacetic acid and 4-hydroxybenzeneacetic acid (Table 4.7; Appendix II i-k).
Naphthalene biotransformation by Bacillus cereus KWS4 resulted the formation of
three metabolites which were detected and identified from the neutral extracts by
GC/MS. The detected metabolites were 2-naphthelenol, benzeneacetic acid and
benzaldehyde (Table 4.8; Appendix III a-c).
From phenanthrene biotransformation by Bacillus cereus KWS4 a total of five
metabolites were detected and identified, three from neutral extracts including; 9phenanthrenol, 9,10-Dihydro, 9,10-dihydroxyphenanthrene and bezeneacetic acid.
While metabolites form acidic extracts were detected and identified as 3-hydroxybenzeneacetic acid, {MS fragment: 152(34), 108(11), 107(100), 86.9(10), 77(22),
63(15), 51(11), 39(6)} and 2-hydroxy-benzoic acid {MS fragment: 137.9(56),
119.9(99), 92(100), 64(29), 63(22), 53(11), 43(23), 39(16)} (Table 4.8; Appendix III
d-h).
61
Chapter4Results
Reduction (%)
4 Days
7 Days
50
45
40
35
30
25
20
15
10
5
0
4
pH
Figure 4.1: Percentage reduction of anthracene concentration (10 ppm) in PNRG

medium at different pH values by Bacillus cereus KWS2 in shake flask experiment.
Figure 4.2: Growth (CFU/mL) of Bacillus cereus KWS2 in PNRG medium with 10
ppm anthracene at different pH values.
62
Chapter4Results
Reduction (%)
4 Days
7 Days
50
45
40
35
30
25
20
15
10
5
0
30
37
45
Temperature
50
Figure 4.3: Percentage reduction of anthracene concentration (10 ppm) in PNRG

medium at different temperature by Bacillus cereus KWS2 in shake flask experiment.
Figure 4.4: Growth (CFU/mL) of Bacillus cereus KWS2 in PNRG medium with 10
ppm anthracene at different temperature.
63
Chapter4Results
Figure 4.5: Percentage reduction of naphthalene concentration by Pseudomonas

stutzeri 10-1.
Figure 4.6: Percentage reduction of anthracene concentration by Pseudomonas

stutzeri 10-1.
64
Chapter4Results
Table 4.7: GC mass spectral data of metabolites from naphthalene, phenanthrene and
anthracene degradation by B. cereus KWS2
Anthracene
Phenanthrene
Naphthalene
Compound Metabolites
Retention
time (min)
m/z of fragment ions (%)

relative abundance
Benzaldehyde
10.24
106(100), 105(96), 78(20),

77(93), 74(13), 52(9),
51(42), 50(24), 39(6)
Benzenecarboxylic acid
13.69
121.9(96), 105(100),
76.9(81), 51(32), 50(25),
43(30)
Benzeneacetic acid
15.02
135.9(31), 92(19), 91(100),

89(5), 65(16), 63(8), 39(7)
2-naphthol
18.75
144(100), 145(12),
116(48),115(95), 89(14),
72(5), 63(10), 50(4), 39(4)
Bezeneacetic acid
15.08
135.9(31), 92(19), 91(100),

89(5), 65(16), 63(8), 39(7)
4-hydroxy-benzeneacetic
acid,
19.35
151.9(33), 108(8), 107(100),

77(23), 51(7), 39(4)
9,10-Dihydro, 9,10dihydroxyphenanthrene
25.18
212(58), 193.9(34), 181(67),

166(51), 165(100), 153(13),
152(24), 138.9(4), 115(6),
82(9), 76.9(10)
9-phenanthrenol
25.48
194(100), 166(33), 165(90),

164(15), 163(16), 139(9),
82(11)
acid
19.35
152(34), 107(100), 108(8),

77(20), 51(6), 39(3)
Benzeneacetic acid
15.25
136(36), 105(100), 101(24),

92(22), 75(9), 65(16), 39(7)
14.04
122(85), 105(100), 77(62),

74(10), 50(16), 39(6)
65
Chapter4Results
Table 4.8: GC mass spectral data of metabolites from naphthalene and phenanthrene
degradation by B. cereus KWS4
Phenanthrene
Naphthalene
Compound
Metabolites
Retention
time (min)
m/z of fragment ions

(%) relative abundance
Benzaldehyde
10.23
106(100), 105(98),
78(22), 77(93), 74(16),
51(38), 50(27)
Benzeneacetic acid
15.02
135.9(34.5), 92(18),
91(100), 65(16.5), 51(5),
39(7.5)
2-naphthol
18.73
144(100), 116(45),
115(98), 89(12.5),
63(10), 50(3.5), 39(4)
Bezeneacetic acid
15.12
135.9(35), 92(19),
91(100), 65(16), 51(5),
39(8)
2-hydroxy-benzoic acid
15.94
137.9(56), 119.9(99),
92(100), 64(29), 63(22),
53(11), 43(23), 39(16)
acid,
19.27
152(34), 108(11),
107(100), 86.9(10),
77(22), 63(15), 51(11),
39(6)
9,10-Dihydro, 9,10dihydroxyphenanthrene
25.19
212(55), 194(34),
181(65), 166(47.5),
165(100), 15.9(26),
115(7.5), 82(8), 77(9)
9-phenanthrenol
25.48
194(100), 166(33),
165(99), 164(14),
163(15.5), 139(9), 82(11)
66
Chapter4Results

Seven different Mycobacterium strains were screened for their growth in BSM
medium supplemented with 0.05% of yeast extract and 10 ppm of pyrene. The result
showed that the Mycobacterium PY146 had a maximum growth after 48 hours with
an O.D600 of 0.65 compared to other strains with O.D600 around 0.5 (Figure 4.7). The
Mycobacterium PY146 strain was selected for further biotransformation studies.
The effect of temperature on the growth of Mycobacterium PY146 was evaluated in
the LB broth. The results showed that the maximum growth was observed at 30C
with O.D600 of 1.12 after 120 hours, and growth decreased at both 25 and 37C
(Figure 4.8).
The effect of pH on growth of Mycobacterium PY146 in LB broth medium was also
evaluated. The maximum growth was observed at pH 7 with an O.D600 of 1.15 after
120 hours. Good growth was also observed at pH 6 with a maximum OD600 around
1.12. There was less growth at pH 5 and 8 (OD600s of 0.4 and 0.6), respectively
(Figure 4.9).
Resting cell biotransformation of PAHs including phenanthrene, anthracene and
pyrene by Mycobacterium PY146 were checked individually. Phenanthrene
biotransformation after 24 hours of incubation resulted in the detection of one
metabolite identified as 1,2-benzenedicarboxylic acid {Mass fragment: 148(23),
105(8), 104(100), 76(68), 74(15), 50(25), 38(6)} (Table 4.9; Appendix IV a) in the
acidic extract of ethyl acetate.
Two metabolites, 1,2-Benzenedicarboxylic acid from acidic extract and 9,10anthracendione {Mass fragment: 209(16), 208(100), 207(15), 181(12), 180(81),
152(61), 151(32), 150(14), 126(7), 76(20), 50(6)} (Table 4.9; Appendix IV b & c)
from neutral extract of ethyl acetate were detected and identified from the anthracene
biotransformation after 24 hours of incubation.
From pyrene biotransformation by Mycobacterium PY146, two metabolites, 1,2Benzenedicarboxylic acid from the acidic and 1-hydroxypyrene {Mass fragment:
219(19), 218(100), 217(12), 190(9), 189(41), 188(4), 187(6), 163(2), 94(3), 43(10)}
67
Chapter4Results
(Table 4.9; Appendix IV d & e) from the neutral extract of the ethyl acetate were
detected and identified.
Growing cell biotransformation of phenanthrene, anthracene and pyrene were also
checked individually by Mycobacterium PY146 in BSM medium with 0.05% yeast
extract in a shaking incubator at 30C.
From the growing cell biotransformation of phenanthrene by Mycobacterium PY146,
1,2-Benzenedicarboxylic acid was detected from the acidic extract of ethyl acetate
from both after 5 and 10 days of incubation, while benzene acetic acid (Table 4.10;
Appendix V a & b) was detected and identified in the acidic extract of ethyl acetate
after 10 days of incubation.
Four metabolites were detected from anthracene by Mycobacterium PY146 growing
cells biotransformation, in which 9,10-anthracenedione from the neutral extract and
benzenecarboxylic acid, benzeneacetic acid and 1,2-benzenedicarboxylic acid ( Table
4.10; Appendix V c - f) from the neutral extract of ethyl acetate after 10 days of
incubation.
Biotransformation of pyrene by growing cells of Mycobacterium PY146 resulted in
three metabolites including benzenecarboxylic acid, benzeneacetic acid and 1,2benzenedicarboxylic acid ( Table 4.10; Appendix V g-i) were detected and identified
from the acidic extract of the ethyl acetate after 7 days of incubation. After 14 days of
incubation one metabolite 1,2-benzenedicarboxylic acid was detected and identified
from the acidic extract of ethyl acetate.
68
Chapter4Results
Figure 4.7: Growth of different Mycobacterium strains on 10 ppm pyrene in BSM

medium.
69
Chapter4Results
Figure 4.8: Effect of different temperature on the growth of Mycobacterium PY146.
Figure 4.9: Effect of different pH on the growth of Mycobacterium PY146.
70
Chapter4Results
Table 4.9: GC mass spectral data of metabolites from phenanthrene, anthracene and
pyrene degradation by resting cells of Mycobacterium PY146
Pyrene
Anthracene
Phenanthrene
Compound
Metabolites
Retention m/z of fragment ions (%)

time (min) relative abundance
acid
17.04
148(23), 105(8), 104(100),

76(68), 74(15), 50(25),
38(6)
1,2Benzenedicarboxylic
acid
17.05
148(23), 104(100), 105(8),

76(66), 74(10), 50(21),
27.2(5)
9,10-anthracendione
25.04
209(16), 208(100),
207(15), 181(12), 180(81),
152(61), 151(32), 150(14),
126(7), 76(20), 50(6)
acid
17.03
148(22), 104(100), 105(8),

76(68), 74(16), 50(22),
43(14), 38.1(7)
1-hydroxypyrene
32.84
219(19), 218(100),
217(12), 190(9), 189(41),
188(4), 187(6), 163(2),
94(3), 43(10)
71
Chapter4Results
Table 4.10: GC mass spectral data of metabolites from phenanthrene, anthracene and
pyrene degradation by growing cells of Mycobacterium PY146
Pyrene
Anthracene
Phenanthrene
Compound
Metabolites
Retention m/z of fragment ions (%)

time (min) relative abundance
Benzene acetic acid
15.14
136(35), 92(20), 91(100),

65(15), 63(6), 51(4), 39(7)
acid
16.04
148(21), 106(9), 104(100),

76(77), 75(11), 74(18),
50(33), 38(7)
13.89
122(84), 105(100), 77(69),

74(11), 73(13), 51(32),
50(21), 43(17)
Benzeneacetic acid
15.2
136(35), 92(20), 91(100),

65(16), 63(9), 51(4), 39(7)
acid
16.03
147.9(19), 105(8),
104(100), 76(70), 74(17),
50(30), 38(6)
9,10-Anthracenedione
23.92
208(99), 180(100),
152(71), 151(40), 150(19),
126(10), 76(29), 50(13)
12.95
122(98), 105(100), 87(26),

77(72), 74(8), 73(17),
72(19), 51(28)
Benzeneacetic acid
14.37
136(34), 92(19), 91(100),

79(8), 65(14), 63(6), 51(5)
acid
15.03
148(21), 105(9), 104(100),

76(74), 75(10), 74(18),
50.1(31)
72
Chapter4Results

Biodegradation of phenanthrene, anthracene and pyrene were checked in soil by
Mycobacterium PY146 with and without additional yeast extract. The extraction
efficiency of the method was 81.6 % for phenanthrene, 83.33 % for anthracene and
77.7 % for pyrene. Figure 4.10 shows the biodegradation of PAHs in soil without
yeast extract, after one week of incubation the detectable concentration of
phenanthrene (98 g), anthracene (60 g) and pyrene (56 g) were decreased to 35,
18 and 27 g, respectively. After 2 weeks of incubation all of the detectable PAHs
were 0.14, 0.45 and 0.19 g respectively.
The addition of yeast extract did not affect the biodegradation of PAHs, except
phenanthrene was detected at 27 g, while anthracene and pyrene were detected 15
and 24 g, respectively after one week of incubation. The detectable PAHs
concentrations of phenanthrene, anthracene and pyrene after 2 weeks of incubation
were 0.08, 0.13 and 0.32 g respectively (Figure 4.11).
73
Chapter4Results
Figure 4.10: Biodegradation of PAHs (phenanthrene, anthracene and pyrene) in soil

by Mycobacterium PY146 without addition of yeast extract.
Figure 4.11: Biodegradation of PAHs (phenanthrene, anthracene and pyrene) in soil

with addition of yeast extract by Mycobacterium PY146.
74
Chapter4Results

4.3.1 14C-PAH mineralization by Bacillus cereus KWS2 and
Mineralization studies of different
14
C-PAHs, including naphthalene, phenanthrene,
pyrene and benzo(a)pyrene were carried out using Bacillus cereus KWS2 and
Mycobacterium PY146 in BSM medium and incubated for one week at 30C.
In case of naphthalene and phenanthrene mineralization by Bacillus cereus KWS2, no
mineralization was observed. In contrast, Mycobacterium PY146 showed the ability to
mineralize the phenanthrene, pyrene and benzo(a)pyrene to CO2. In the case of
phenanthrene,a total of 77.32 % radiolabeled 14C was recovered, 45.92% of 14C was
detected in the form of Na214CO3, 23.19 % of the 14C remained in the supernatant and
8.21 % of the
14
C was detected in the biomass/residues (Figure 4.12). In the case of
pyrene mineralization, a total of 65.77 % radiolabeled 14C was recovered, 39.89 % of

14
C was detected in the form of Na214CO3, 15.18 % of the14C remained in the
supernatant and 10.7 % of the 14C was detected in the biomass/residues (Figure 4.13).
In the in case of benzo(a)pyrene mineralization, a total of 42.84% radiolabeled
was recovered, 4.82 % of
14
14
C was detected in the form of Na214CO3, 22.32 % of
the14C remained in the supernatant and 15.7 % of the
14
C was detected in the
biomass/residues (Figure 4.14).
4.3.2 14C-Pyrene and phenanthrene mineralization using with

The mineralization of pyrene by Mycobacterium PY146 was performed using
incubation times ranging from 4 to 48 hours. Little mineralization occurred in the first
5 hours of incubation but after 20 hours of incubation, 37.9 % of the pyrene was
mineralized and by 48 hours 100 % of the pyrene was mineralized (Figure 4.15). The
middle incubation time of 20 hours was selected for further studies.
The mineralization of pyrene with different cell densities of Mycobacterium PY146
was performed in 40 ml EPA vials. After 20 hours of incubation, the maximum
mineralization was observed in the OD600 0.2 of culture. The mineralization pattern
showed an increase in mineralization in the OD600 0.1 to 0.2 cultures with a
75
Chapter4Results
subsequent decrease in mineralization as the OD600 increased.
The minimum
mineralization was observed by the culture having a cell density of 0.5 (Figure 4.16).
Mineralization of phenanthrene was also evaluated using different Mycobacterium
PY146 cell densities ranging from an OD600 of 0.1 to 0.5. The mineralization pattern
also showed an increase in mineralization between the OD600 0.1 to 0.2 culture where
maximum mineralization was observed. There also was a subsequent decrease in
mineralization as the cell density increased (Figure 4.17).
76
Chapter4Results
Figure 4.12: 14C-phenanthrene mineralization by Mycobacterium PY146.
Figure 4.13: 14C-pyrene mineralization by Mycobacterium PY146.
77
Chapter4Results
Figure 4.14: 14C-benzo(a)pyrene mineralization by Mycobacterium PY146.
Figure 4.15: Mineralization of

different incubation time.
14
C-pyrene by cells of Mycobacterium PY146 at
78
Chapter4Results
60000
Mineralization of Pyrene (DPM)
50000
OD vs DPM
40000
30000
20000
10000
0.0
0.1
0.2
0.3
0.4
0.5
0.6
O.D (600 nm)
Figure 4.16: Mineralization of pyrene with different cell densities by Mycobacterium

PY146.
Mineralization of Phenanthrene (DPM)
70000
60000
OD vs DPM
50000
40000
30000
20000
10000
0.0
0.1
0.2
0.3
0.4
0.5
0.6
O.D (600 nm)
Figure 4.17: Mineralization of

14
C-phenanthrene with different cell densities by
79
Chapter4Results

FROM MYCOBACTERIUM PY146
4.4.1 Cloning of nidAB gene
The strain Mycobacterium PY146 was grown in LB medium for 72 hours, the
genomic DNA was isolated and used as a template for the amplification of nidAB
gene. After PCR amplification with nidAB primers, an approximately 1.9 Kb
amplicon size of nidAB was detected when the PCR products were run on a 1 %
agarose gel (Figure 4.18).
After the nidAB PCR product was ligated into a pCR 2.1 TOPO vector, 6 L of
recombinant nidAB- pCR 2.1 TOPO vector was added to a vial of competent E.coli
cells for transformation. Transformed E. coli cells were grown on LB agar plates
containing 100 g/mL ampicillin. Single colonies were regrown in LB broth
containing ampicllin and the plasmid DNA was isolated. After digestion with EcoR1,
the isolated plasmid DNA from three samples had both the plasmid backbone
fragment (3.9 Kb) and cloned nidAB gene fragment (1.9 Kb) (Figure 4.19).
Restriction sites for EcoR1 and HindIII were incorporated into both ends of the
nidAB by amplification using the primers nidABEco forward and nidABHind reverse.
The nidAB gene with EcoR1 and HindIII restriction sites was cloned into the TOPO
vector and transformed into competent E. coli cells.
Following plasmid DNA
extraction, the plasmid was sequenced using the M13 forward and reverse primers to
confirm the presence of the EcoR1 and HindIII restriction sites at the ends of nidAB
gene (Appendix I c).
The TOPO cloning vector containing the EconidABHind product and pK233-3
expression vector were digested with EcoRI and HindIII and purified. Following
ligation of the EconidABHind with pK223-3, the plasmid products were transformed
into E. coli and E. coli containing functional plasmids was selected using ampicillin
antibiotics in the medium. Plasmid DNA was isolated from E. coli and restriction
enzyme analysis of isolated plasmid DNA with EcoR1 and HindIII revealed the
presence of both the fragments of pK223-3 vector and nidAB in 6 out of 8 samples
80
Chapter4Results
(Figure 4.20). Direct colony PCR from the transformed E. coli cells with nidAB
primers confirmed the presence of the cloned nidAB gene (Figure 4.21).
4.4.2 Biotransformation of PAHs by E. coli with cloned nidAB into pkk223-3

Resting cell biotransformation of naphthalene, phenanthrene, anthracene and pyrene
was performed using E. coli cells containing nidAB into pkk223-3 grown in LB
medium and with each chemical added individually in shake flask experiments. After
extraction with ethyl acetate and analysis on GC/MS, no hydroxy metabolites of any
of the PAH were detected.
In addition, no metabolites were detected for any of the PAH in GC/MS analysis after
derivatization
with
N,O-Bis(trimethylsilyl)trifluoroacetamide
with
Trimethylechlorosilane (BSTFA).

RNA transcripts of nidAB were synthesized invitro using the T7 RiboMAX
Express Large Scale RNA Production System from the TOPO plasmid. Following
invitro transpcription, the concentration of nidAB RNA was 95.3 ng/L as measured
by the nanodrop spectrophotometer . This RNA was used as a standard for RT-QPCR.
4.4.3.1 Quantification of RNA transcripts of nidAB from E. coli

For the expression of nidAB gene, the transformed E. coli cells (clone numbers 6, 7
and 8 containing pKK223-3) were grown in LB medium containing ampicillin with
and without IPTG and the RNA were isolated. The RNA copy number/ml of bacterial
culture were quantified by RT-QPCR which revealed an ~ 2- fold increase in RNA
transcripts in one sample grown in the presence of IPTG (Figure 4.22).
4.4.4 Comparison of nidAB DNA and RNA copy number using realtime PCR with PAH mineralization in Mycobacterium PY146
Total DNA and RNA isolated and purified from Mycobacterium PY146 were
quantified by using real time PCR. Both nidAB genes (DNA) and transcripts (RNA)
increased with increasing OD600 (Figure 4.23). An RNA to DNA ratio also calculated
81
Chapter4Results
which showed that the RNA to DNA ratio increased between OD600 0.1 to 0.4 and
then declined at an OD600 of 0.5 (Figure 4.24). In order to relate mineralization
activity with nidAB RNA transcripts, phenanthrene mineralization was compared to
the RNA/DNA ratio. These results indicated that there was a positive relationship
between phenanthrene mineralization and the RNA/ DNA ratio (Figure 4.25).
Comparison of pyrene mineralization with and without added unlabelled pyrene, with
the nidAB RNA/DNA ratio by different cell concentration indicated that the nidAB
RNA/DNA ratio only corresponded to the amount of pyrene mineralization between
an OD600 of 0.3 to 0.5 (Figure 4.26).
82
Chapter4Results
1938bp
Figure 4.18: Ethidium bromide stained agarose gel showing the PCR product of
nidAB gene from Mycobacterium PY146. Lane L: 1KB DNA ladder; Lanes 1: nidAB.
The arrow indicates the location of the nidAB product.
L
~ 3.9 Kb
~ 1.9 Kb
Figure 4.19: Ethidium bromide stained 1 % agarose gel showing the location of the
plasmid (top arrow) and nidAB gene (bottom arrow) after digestion with EcoR1. Lane
L: 1KB DNA ladder; Lanes 1, 2 and 5: have both the plasmid and nidAB.
83
Chapter4Results
L 1
Figure 4.20: Ethidium bromide stained 1 % agarose gel showing the plasmid and
nidAB after digestion with EcoR1 and HindIII. Lane L: 1KB DNA ladder; Lanes 1
and 2 have only plasmid; Lanes 3-8: have both the plasmid and nidAB.
L
~ 1.9 Kb
Figure 4.21: Ethidium bromide stained 1 % agarose gel showing the presence of
cloned nidAB gene in transformed E. coli. Lane L: 1KB DNA ladder; Lanes 1 to 6: all
have the cloned nidAB.
84
Chapter4Results
Figure 4.22: Copies of nidAB RNA transcripts in E. coli clones grown in LB

containing ampicillin with and without IPTG.
85
Chapter4Results
Copies DNA or RNA/ml of Bacteria
1e+10
DNA copies/ml (r2 =0.90)

RNA copies/ml (r2= 0.92)
1e+9
1e+8
1e+7
0.0
0.1
0.2
0.3
0.4
0.5
O.D at 600 nm
Figure 4.23: Standard curves of DNA and RNA/ml of bacteria from different cell
densities from 0.1 to 0.5 O.D.
2.8
2.6
RNA to DNA Ratio
2.4
Ratio RNA/DNA
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.0
0.1
0.2
0.3
O.D at 600 nm
0.4
0.5
Figure 4.24: RNA to DNA ratio at different cell densities from 0.1 to 0.5 O.D.
86
Chapter4Results
2.8
2.6
60000
2.4
50000
2.2
40000
2.0
1.8
30000
1.6
RNA/DNA Ratio
Mineralization of Phenanthrene (DPM)
70000
20000
1.4
OD vs Phenanthrene
OD vs ratio
10000
1.2
1.0
0.0
0.1
0.2
0.3
0.4
0.5
0.6
O.D at 600 nm
Figure 4.25: Comparison of mineralization of phenanthrene from different cell

densities and the nidAB RNA/DNA ratio in Mycobacterium PYR146.
1e+5
2.8
2.4
1e+4
2.2
2.0
1.8
1e+3
1.6
OD vs DPM without add pyrene
OD vs DPM with add pyrene
OD vs ratio
RNA/DNA Ratio
Mineralization of Pyrene (DPM)
2.6
1.4
1.2
1e+2
1.0
0.0
0.1
0.2
0.3
0.4
0.5
O.D at 600 nm
Figure 4.26: Effect of added un-labled pyrene on the mineralization of 14C- labled
pyrene at different cell densities of Mycobacterium PYR146 and the nidAB
RNA/DNA ratio.
87
Chapter5Discussion
Polycyclic aromatic hydrocarbons (PAHs) are a ubiquitous class of organic
compounds present in the environment. Microbial degradation of PAHs by bacteria
and fungi is considered a safe and environment friendly method to remove toxic
contaminants. In the present study, bacterial isolates utilizing the PAHs (naphthalene,
anthracene, phenanthracene, and pyrene as sole carbon and energy were characterized
taxonomically and for their degradation potential for the respective PAH.
Soils contaminated with hydrocarbon are good sources for the isolation of PAHs
degrading bacteria (Jacques et al., 2009; Al-Thani et al., 2009) which can then be used
for the removal of such compounds from the environment. In this context, the
bacterial strains in this study which were isolated from the crude oil contaminated soil
utilized polycyclic hydrocarbons as sole source of carbon energy. Among these five
selected bacterial strains, KWS2, KWS4, DW3, Sol-10 and 10-1 showed the ability to
tolerate the PAHs concentration up to 1200 ppm in case of anthracene and 800 ppm in
case of pyrene (Table 4.1 & 4.2). These selected isolates were identified as; Bacillus
cereus, Bacillus licheniformis and Pseudomonas stutzeri (Table 4.6). Previously
different strains of Bacillus have been isolated from PAHs contaminated soil (Jacques
et al., 2009; Das and Mukherjee, 2007; Lin et al., 2010), which have the potential to
biodegrade and utilize organic compounds like phenanthrene, anthracene,
naphthalene, biphenyl and some other aromatic compounds. Thermophillic bacteria
belonged of genus Geobacillus have also been reported by Bubinas et al., (2008)
capable of utilizing naphthalene as a sole source of carbon. Pseudomonas species
have been well studied for their ability to degrade the different type of pollutants
including; PCBs (De et al., 2006), textile dye direct orange 102 (Pandey et al., 2010)
and crude oil components (Tang et al., 2007). Pseudomonas species have also been
reported in several studies for their degradation potential of PAHs from naphthalene
to benzo(a)pyrene
isolated from different PAHs contaminated soils. Coral and
Karagoz, (2005) isolated several Pseudomonas strains from crude oil polluted soil of
a petroleum refinery which degraded phenanthrene efficiently. Pseudomonas species
also were isolated from soil samples from located at a petrochemical treatment plants
showed degradation capability toward different PAHs (Jacques et al., 2009; Molina et
al., 2009; Aitken et al., 1998). The isolation of bacterial strains in the present study
from oil contaminated soil suggested that these isolates may be well adapted for use
88
Chapter5Discussion
because of pre-exposure and acclimatization to the contaminants and can be used
effectively in remediating the contaminated sites.
In biodegradation studies of anthracene by Bacillus cereus KWS2, a 45 % reduction
in anthracene concentration was observed at pH 7 and 30C after seven days of
incubation (Figure 4.1). These results are consistent with other reports on the
efficiency of Bacillus to degrade PAHs. For instance, Bacillus fusiformis isolated
from wastewater sludge of an oil refinery showed optimal naphthalene degradation at
pH 7 and 30C (Lin et al., 2010). Shuyu et al., (2007) reported the isolation of two
bacterial strains identified as Bacillus sp. having the ability to degrade pyrene
individually as well as in consortium and optimally at 37C at pH 7. A novel strain of
Bacillus subtilis which can utilize several PAHs including benzo(a)pyrene,
anthracene, naphthalene and dibenzothiophene as sole source of carbon energy was
isolated from a contaminated soil at an automobile workshop (Lily et al., 2009). The
pH of the medium affect the degradation rate, as Kim et al., (2005) suggested that
PAHs at pH 6.5 can get enter to the bacterial cell rapidly comparing to pH 7.5. The
degradation of naphthalene and anthracene by Pseudomonas stutzeri 10-1 was
45.35% reduction for naphthalene (in two weeks) (Figure 4.5) while 95.56 %
reduction in anthracene (three weeks time) concentration (Figure 4.6) was achieved.
Pseudomonas stutzeri P-16 and P. saccharophila P-15 isolates from creosote
contaminated soil were reported to degrade naphthalene, 2-methylnaphthalene and 1methylnaphthalene (Stringfellow and Aitken, 1995). Maximum degradation of
naphthalene by Pseudomonas sp. HOB1 was achieved in the pH and temperature
ranges of 7.5-8.5 and 35-37C, respectively (Pathak et al., 2009). The significant
reduction in total petroleum hydrocarbon (TPH) concentration in the soil after
treatment was reported by a consortium of Bacillus subtilis and two strains of
Pseudomonas aeruginosa (Das and Mukherjee, 2007).
For biodegradation the microbial potential is very critical due to the presence of
catabolic genes on plasmid and chromosomes enable them to degrade and utilize as
carbon source. In case of PAHs the compounds are not easily available, some bacteria
have the potential to produce enzymes and biosurfactant to solubilize the compounds
and make available for the degradation.
89
Chapter5Discussion
In addition to microbial potential, the right ecological conditions are also very
important for microbial activity for biodegradation. The temperature, pH, oxygen and
nutrients availability often affect the rate of biodegradation. Temperature and pH of
the medium affects the PAHs degradation by increasing the solubility of the
compound (Lau et al., 2003) and also the optimum temperature enhances the
microbial activity. As the aerobic metabolism of PAHs is efficient than anaerobic,
oxygen is required for mono and dioxygenases for the initial oxidation of PAHs.
Inorganic nutrients serve always as limiting factor and required for the microbial
degradation and growth on PAHs. The studies showed the enhanced degradation rates
of PAHs when potassium, nitrogen and phosphorous were added to the medium
(Hwang et al., 1994).
A variety of catabolic mechanisms including role of different oxygenases have been
reported in Bacillus regarding biodegradation of monocyclic aromatics and PAHs.
Such metabolic transformation abilities of Bacillus were also evaluated in the present
study by using Bacillus cereus strains KWS2 and KWS4. These results proved that
these isolates were capable of transforming and degrading naphthalene, phenanthrene
and anthracene to metabolites when observed through GC/MS. Metabolites were
detected in GC/MS chromatograph analysis when naphthalene was used as growth
substrate in resting cells of B. cereus KWS2 and B. cereus KWS4. These were 2naphthol, benzeneacetic acid, 2-hydroxy benzeneacetic acid, benzenaldehyde and
benzoic acid (Fig 5.1). One metabolite identified as 2-naphthol which have been
described as intermediate of naphthalene degradation by B. cereus (Cerniglia et al.,
1984) and Bacillus thermolrovanas (Annweiler et al., 2000; Bubinas et al., 2008). oPhthalic acid was not detected in our study but was confirmed as transformation
metabolites of 1-naphthol or 2-naphthol in studies with Geobacillus (Bubinas et al.,
2008). Benzoic acid was detected as metabolite of naphthalene degradation by B.
cereus KWS2 and KWS4 (Fig 5.1) which is also reported in the naphthalene
degradation by B. thermoleovorans (Annweiler et al., 2000).
Metabolites accumulating during resting cells incubation with three ring compound
phenanthrene with B. cereus KWS2 and B. cereus KWS4 showed monoxygenase
activity and dioxygenase activity as mono-hydroxy phenanthrene and cis-dihydroxy90
Chapter5Discussion
dihydro phenanthrene were detected showing attack on C9 and C10 position.
Degradation pathway described by Staphylococcus sp. PWY was showing initial
attack at C1 and C2 position giving 1,2-dihydroxy phenanthrene (Mallick et al.,
2007). Further metabolism of 9,10 dihydro, dihydroxy phenanthrene to 3-hydroxy
benzeneacetic acid, 2-hydroxy benzeneacetic and 4-hydroxy benzeneacetic acid
(Figure 5.1) showing ring cleavage and hydroxylation again by monoxygenase attack.
Phenanthrene trans 9-10 dihydrodiol was also detected in the growth medium of
Mycobacterium strain grown on commercial anthracene (Tongpim and Pickard, 1999)
and by Streptomyces flavovires (Sutherland et al., 1990). Luan et al., (2006) also
reported a mono and dihydroxy phenanthrene from phenanthrene degradation by a
bacterial consortium. Other gram negative bacteria known to oxidize anthracene by
dioxygenase to anthracene cis-1,2-dihdrodiol (Cerniglia and Heitkamp, 1989), while
gram positive bacteria like Coryneform bacillus (Bouchez et al., 1996) also degraded
anthracene but no metabolites were identified.
In the present study, 2-hydroxy benzoic acid (salicylic acid) was detected in the
phenanthrene biotransformation by B. cereus KWS4 (Figure 5.1). Our finding
confirms those reported by Mallick et al., (2007) who identified the salicylic acid
from phenanthrene biotransformation by Staphylococcus sp. PN/Y and proposed its
further transformation to catechol.
Over the last decade the genus Mycobacterium has been proved to be one of the best
bacterial types involved in PAHs degradation. In this context a number of
Mycobacterium species having PAHs degradation capabilities have been isolated
from river and fresh water sediments (Kageyama et al., 2007; Churchill et al., 1999).
Their metabolic successes have been associated with their cell wall constituent
mycolic acid which provides great resistant abilities to withstand recalcitrant
hydrocarbons which later help them to metabolize those compounds. Moreover, the
hydrophobic nature of mycolic acid helps developing biofilms on surface of PAHs,
which can later be help promote community biodegradation (Wick et al., 2002a).
Mycobacterium sp. has high specific affinity for PAHs and grows on the surface of
solid anthracene when provided in low amounts, attached to the crystal and form
biofilm and use as a carbon source (Wick et al., 2002b). The present study has also
used strain of Mycobacterium PY146 previously isolated from PAH contaminated
91
Chapter5Discussion
sediment of Lake Erie (Debruyn et al., 2007). This strain showed 98% homology with
Mycobacterium vanbaalenii PYR-1 which efficiently degrades and mineralizes
pyrene and phenanthrene in liquid medium as well as in microcosms. Phenanthrene
and pyrene degradation pathways also have been constructed for this strain (Heitkamp
and Cerniglia 1989; Heitkamp et al., 1988a,b; Stingley et al., 2004a,b; Kim et al.,
2004).
In PAHs biotransformation studies, the presence of metabolites in the medium
suggested that Mycobacterium PY146 has the ability to degrade phenanthrene,
anthracene and pyrene efficiently. 1,2-benzenedicarboxyic acid, also known as
phthalic acid, was detected as degradative metabolite from all the three compounds
tested.
Pyrene metabolism by Mycobacterium PY146, identified an 1-hydroxy
pyrene, showing momooxygenase attack. In addition 9, 10-anthracenedione was

detected as a metabolite from anthracene and 1-hydroxypyrene from pyrene
degradation (Table 4.9 & 4.10; Figure 5.2). Phthalic acid was also detected in all the
phenanthrene, anthracene and pyrene. Phthalic acid has also been reported in pyrene
metabolism by Mycobacterium sp. AP1 (Vila et al., 2001). Phthalic acid is also
produced in the degradation pathway of phenanthrene by bacterial consortium (Luan
et al., 2006). It is known that phthalic acid is further metabolized in aerobic bacteria
through protocatecuate as intermediate (Grifoll et al., 1994).
Phthalic acid from phenanthrene and 9,10-anthraquinone from anthracene
biodegradation along with other metabolites by Mycobacterium PYR-1 were also
reported by Moody et al., (2001). Dean-Ross and Cerniglia, (1996) reported the
phthalic acid and other metabolites from pyrene by Mycobacterium flavescens. The 1hydroxypyrene detection from pyrene biotransformation by Mycobacterium PY146 in
the present studies suggests a monoxygenase activity. Brenza et al., (2006) reported
the presence and activity of cytochrome P450 monoxygenase gene in Mycobacterium
vanbaalenii PYR-1 and detected a 1-hydroxypyrene from pyrene degradation. Zhong
et al., (2006) also reported the presence of both mono and di-hydroxypyrene
metabolites from pyrene degradation, suggested that the PAH molecule metabolized
through mono and dihydroxylation by Mycobacterium sp. strain A1-PYR.
92
Chapter5Discussion
Biodegradation of PAHs in soil by microbial action is of great importance because
soil and sediment is a sink for the accumulation of hydrophobic pollutants especially
polycyclic aromatic hydrocarbons. Potentially, the rate of biodegradation could be
changed by the addition of some nutrients. In the biodegradation studies of PAHs;
phenanthrene, anthracene and pyrene in soil revealed the removal of detectable
compounds by Mycobacterium PY146 in two weeks at pH 7 and at 30C (Figure
4.10). The addition of yeast extract did not have a significant effect on biodegradation
of PAHs in our study except on phenanthrene degradation after one week of
incubation (Figure 4.11).
Chang et al., (2001) studied the potential of a microbial consortium in the soil to
degrade phenanthrene. Phenanthrene was degraded efficiently at pH 7 and at 30C and
the degradation rate was enhanced by the addition of yeast extract and, glucose. Wan
et al., (2003) reported the removal of PAHs from spiked soil by composting and
reported a 90 % degradation of anthracene, phenanthrene and pyrene after 30 days.
The addition of pig manure slightly enhanced biodegradation soybean and sewage
sludge. Biodegradation of PAHs (phenanthrene and pyrene) also was studied in a soil
slurry reactor using an immobilized bacterial culture of Zoogloea. . This study showed
an overall degradation of pyrene and phenanthrene was 75.4 and 87 % respectively.
(Li et al., 2005). Wang et al., (2006) used immobilized bacterial consortium to
degrade the PAHs pyrene and benzo(a)pyrene in contaminated soil with a degradation
efficiency of 43.49 and 38.55 % respectively in 96 hours. Su et al., (2007) studied the
ability of three bacterial isolates Bacillus sp. SB02, Zoogloea sp.SB09, and
Flavobacterium sp.SB10 individually to degrade pyrene and benzo(a)pyrene in soil.
All three isolates degrade the PAHs, Bacillus sp. SB02 degrade 42.69 and 33.04 % of
pyrene and benzo(a)pyrene, respectively with the highest rate. The results of
biodegradation of PAHs in soil in present studies is similar and in accordance with the
data presented here from the different studies, while the effect of additional nutrients,
yeast extract had no significant effect on the overall biodegradation process, the
similar results reported by Juhasz et al., (2000) that the addition of yeast extract on the
biodegradation of high molecular weight PAHs had no effect but only affect
positively on low molecular PAHs biodegradation. The supplement of additional
carbon source such as glucose reduces the degradation of PAHs probably due to the
93
Chapter5Discussion
availability of easily degradable carbon by Burkholderia cocovenenans (BU-3)
(Wong et al., 2002) and glucose supplementation also reduced the PAHs dioxygenase
activities (Tiana et al., 2003).
The ultimate goal of biodegradation of organic pollutants is mineralization of the
problematic compound into inorganic constituents such as CO2 and H2O, because
partially degraded compounds often have more toxic effects than their parent
compounds. Mineralization of different PAHs by Bacillus cereus KWS2 and
Mycobacterium PY146 was evaluated.
Bacillus cereus KWS2 was not able to
mineralize the tested PAHs including naphthalene and phenanthrene. Mineralization

was observed for Mycobacterium PY146 and 14CO2 was evolved from phenanthrene,
pyrene and benzo(a)pyrene with yields of 45.92, 39.89 and 4.82 %, respectively
(Figure 4.12; 4.13 & 4.14). Naphthalene was not mineralized by Mycobacterium
PY146. The mineralization results from Mycobacterium PY146 were consistent with
those reported by Moody et al., (2001) which showed the mineralization capability of
Mycobacterium PYR-1 to be 45 and 52 % for anthracene and phenanthrene,
respectively. In another study, Sho et al., (2004) showed that Mycobacterium S65,
isolated from jet fuel contaminated site, was able to mineralize fluoranthene,
phenanthrene and pyrene but not fluorene, anthracene and naphthalene. Dean-Ross
and Cerniglia (1996) also showed that Mycobacterium flavescens could mineralize
phenanthrene, pyrene and fluoranthene but not other PAHs such as naphthalene,
acenaphthalene, anthracene, chrysene, fluorene and benzo(a)pyrene. Finally,
Mycobacterium strain CH1 has the ability to mineralize fluoranthene, phenanthrene
and pyrene, but was unable to mineralize anthracene, fluorene and naphthalene
(Churchill et al., 1999). These results suggest that all Mycobacterium sp. including
Mycobacterium PY146 show selective preference for mineralization of some PAHs.
Sho et al., (2004) suggested that the angularity of the PAH is the limiting factor, and
since naphthalene and anthracene are linear in structure the initial attack of the
microbes on PAH is in the K and bay region of the compound. Sundberg et al., (1997)
also suggested the bacterial attack on bay region is very common because of the open
space of the exposed carbon atoms of PAHs in this region. The intradiol cleavage at K
region diol was also reported by Rhemann et al., (1988) and Vila et al., (2001). The
Mycobacterium is very selective in the initial oxidation of PAHs. M. vanbaalenii
94
Chapter5Discussion
PYR-1 initially attacked the pyrene and phenanthrene nucleus in the 4,5 and 9,10
carbon positions, the K region of each PAH. The oxidation of PAHs at the K-region
bonds seems to be unique to Mycobacterium species (Moody et al., 2004).
Cell density and inoculum age may affect microbial metabolic activity. Thus
mineralization of
14
C pyrene and phenanthrene was evaluated using different cell
densities ranging from OD600 0.1 to 0.5 of Mycobacterium PY146 cultures. Maximum
mineralization of pyrene was observed in the culture with an OD600 of 0.2 (Figure
4.16), while in the case of phenanthrene there was an increase in mineralization in
cultures with an increasing OD600 from 0.1 to 0.2 (Figure 4.17). There were decreases
in both pyrene and phenanthrene mineralization as the cell density increased. Kanaly
et al., (2002a,b) used a microbial consortium isolated from soil to mineralize
benzo(a)pyrene and reported a positive effect on mineralization of BaP after the
addition of diesel fuel and with increasing inoculums sizes up to 10 fold, decreases
the lag phase and a little increase in the mineralization but not what they expected to
be more active mineralization. Ye et al., (1996) reported an effect of cell density of
Sphingomonas paucimobilis on the mineralization of different PAHs with an increase
in mineralization increases with an increase in cell density of the inoculums. Shin et
al., (2005) studied the effect of bacterial growth on phenanthrene degradation and
reported the positive effect of increasing growth on degradation. Semple et al., (2006)
studied the effect of additional inoculum on phenanthrene mineralizing Pseudomonas
and the addition of
14
C-phenanthrene on the mineralization of radio labeled
phenanthrene and suggested the additional phenanthrene was mineralized efficiently

but the addition of bacterial cells did not increase mineralization. The results are not
in accordance with the most of studies which showed the mineralization increases
with the increase in cell density. In our studies the maximum mineralization of PAHs
was observed by the lower cell densities cultures from OD600 0.1 to 0.2, which were
the optimal cell densities. As from the data of the growth curve of Mycobacterium
PY146 (Figure 4.7) the mid log phase is 0.2-0.3 where the cells are highly active
metabolically, and the maximum regulation of catabolic genes occurs.
Aerobic microbial degradation of PAHs is mediated by several different oxygenase
enzymes, including mono and dioxygenase. In some cases, the expression of catabolic
genes can be studied by transferring the gene into a heterologous host like E. coli
95
Chapter5Discussion
(Kurkela et al., 1988; Laurie and Lloyd Jones, 1999 and Kasuga, 2001). For instance,
Sphingomonas LH128, isolated from a PAHs contaminated soil has a dioxygenase
complex, phnA1fA2f, responsible for oxidation of different PAHs. When this gene
was cloned and over expressed in E. coli, the phnA1fA2f gene was shown to confer
the ability to oxidize a different range of PAHs from low molecular weight dibenzo-pdioxin and chlorinated biphenyls to high molecular weight pyrene, chrysene, and
benz(a)anthracene (Schuler et al., 2009). Similarly, the pyrene dioxygenase gene,
nidAB which is encoded by nidA and B ( and subunits) in Mycobacterium is
responsible for the ring hydroxylation of pyrene, which is the initial step in pyrene
metabolism (Khan et al., 2001). In this study, the attempts were carried to express the
pyrene dioxygenase gene (nidAB) from Mycobacterium PY146 in E. coli by cloning
it in an expression vector followed by transforming E. coli. The biotransformation of
naphthalene, phenanthrene, anthracene and pyrene was checked using E. coli cells
containing nidAB, but no hydroxy metabolites or derivatized metabolite were
detected.
These results differ from Khan et al., (2001) who demonstrated the
functionality of the dioxygenase of Mycobacterium PYR-1 after cloning it into E. coli.

They reported the expression of nidAB, carried on a plasmid and nidABD, carried on
a phagemid both individually by transforming the pyrene to pyrene-4,5-dihydrodiol
without reductase and ferrodoxin components. They suspected that this reaction
occurred because the cells were borrowing the reductase and ferredoxin components
from elsewhere in the cell to compliment the NidAB dioxygenase subunits. These
reductase and ferrodoxin are required by the dioxygenase systems similar to nidAB
for the electron transfer (Treadway, et al., 1999). From present study, the possible
cause of the failure in expression of the nidAB gene in E. coli may be due to the lack
of the reductase and ferrodoxin components required for the electron transfer in the
dioxygenase system. This idea is supported by Kim et al., (2006) who studied the
expression of nidA3B3 from Mycobacterium PYR-1in transformed E. coli and found
that no PAH degradation activity was observed until they introduced another plasmid
carrying the ferrodoxin reductase and ferrodoxin from Nocardioides sp. KP7 into the
E. coli. They verified the coexpression of the genes in E. coli using several PAHs
including; naphthalene, anthracene, phenanthrene, fluoranthene, pyrene and
benz(a)anthracene which were all converted to the expected cis-dihydrodiol
metabolites.
96
Chapter5Discussion
The PAH degrading microbial population can be estimated in environmental samples
by quantifying the specific catabolic gene and its activity by quantifying the RNA
transcripts and determining the RNA to DNA ratio. In our study we used the nidAB
RNA transcript to DNA ratio calculated using RT-QPCR for different cell densities of
Mycobacterium PY146. The RNA transcript to DNA ratio increased as the cell
density increased from OD600 0.1 to 0.4 and then declined when the OD600 reached 0.5
(Figure 4.24). The relationship of phenanthrene and pyrene mineralization to the RNA
transcript to DNA ratio was positive for phenanthrene between OD600 0.1 to 0.5
(Figure 4.25), but was only positive for pyrene between OD600 of 0.3 to 0.5 (Figure
4.26). Genes with catabolic activities, such as pyrene dioxygenase nidAB not only
play an important role in the contaminant removal but also serve as markers for the
degradation potential. In our studies the use of RNA transcript to DNA ratio of nidAB
to correlate with the PAHs mineralization gave the clear indication in case of
phenanthrene and pyrene mineralization capability of the bacterial isolate.
Sanseverino et al., (1993) used DNA hybridization with the nahA gene probe,
naphthalene lux reporter system and the messenger RNA transcript of nahA gene to
study PAHs degradation and the bacterial population in soil. They reported a positive
correlation of nahA transcript with the mineralization rate of naphthalene as well as
with the densities of naphthalene degrading microbes determined by colony
hybridization. Park and Crowely, (2006) reported a thousand fold increase in gene
copy number of nahAc when cells were exposed to naphthalene. They also observed
the direct relationship between the gene copy number and the naphthalene
degradation rate in soil contaminated with PAHs. Fleming et al., (1993) studied the
expression of nahA catabolic gene in soil contaminated with PAHs. They also found a
positive correlation of nahA transcript with the naphthalene mineralization and nahA
gene frequency. Tuomi et al., (2004) correlated nahAc gene abundance with the
aerobic
14
C-naphthalene mineralization, total microbial cell counts, respiration rate
and organic matter content in oxic and anoxic soil samples, they found nahAc gene
abundance was correlate with the
14
C-naphthalene mineralization in the oxic soil
layers and the total microbial cell counts were correlated with nahAc gene abundance
in both anoxic and oxic layer of soil. The results of the present study are similar to
these other studies with respect to phenanthrene in that the RNA to DNA ratio of the
97
Chapter5Discussion
nidAB relates with the mineralization of phenanthrene. However, in the case of
pyrene mineralization, the RNA transcript to DNA ratio did not relate to pyrene
mineralization. That may be due the possible activity of the monoxygenase in the
Mycobacterium PY146 in addition to the nidAB as seen from the detection of 1hydroxyryrene metabolites from pyrene degradation.
In this study, the degradation potential, including transformation and mineralization,
were characterized in different isolates from different genera and geographical
locales. From the study it is summarize, that the isolate Bacillus cereus KWS2 and
KWS4 has the potential to degrade and transform different PAHs but could not
mineralize them. In contrast, Mycobacterium PY146 has both the degradation and
mineralization potential for most of the studied PAHs. The expression of pyrene
dioxygenase nidAB in a heterologous system requires the ferrodoxin and reductase
components which was not present when the nidAB was cloned into E. coli. It is also
suggested that monoxygenase along with the nidAB dioxygenase is present and active
in the Mycobacterium PY146. Therefore the use of these isolates alone or in
combination could result in effective degradation of PAHs and related compounds in
the bioremediation process in the contaminated environment.
98
Chapter5Discussion
Naphthalene
Phenanthrene
OH
OH
HO
2-naphthol
OH
9-phenanthrenol
HO
9,10 Dihydro 9,10 dihydroxyphenanthrene

Benzeneacetic acid
OH
OH
O
O
HO
HO
HO
Benzeneacetic acid
Phthalic acid
Phthalic acid
HO
HO
HO
(Benzoic acid)
2-hydroxy benzoic acid

(Salicylic acid)
HO
HO
HO
HO
Catechol
Catechol
Benzaldehyde
Figure 5.1: Proposed pathway for the degradation of naphthalene and phenanthrene by
B. cereus KWS2 & KWS4. Compounds in brackets are from other reported studies.
99
Chapter5Discussion
Anthracene
Phenanthrene
Pyrene
OH
O
O
HO
HO
1,2 benzenedicarboxylic acid
9,10 anthracendione
1, hydroxypyrene
O
OH
OH
HO
Benzeneacetic acid
HO
HO
HO
HO
O
O
HO
Benzeneacetic acid
HO
Benzeneacetic acid
Figure 5.2: Metabolites detected from the biotransformation of phenanthrene,

anthracene and pyrene by Mycobacterium PY146.
100
Conclusions
Conclusions
PAHs contaminated soil is the rich source of PAH degrading microbes.
Bacillus was found to be the major contributor amongst the isolated bacterial
strains from Pakistan.
From the GC/MS data Bacillus cereus KWS2 and B. cereus KWS4 are able to
transform naphthalene, phenanthrene and anthracene.
The GC/MS and mineralization data showed that, the Mycobacterium PY146
degrades phenanthrene, anthracene, pyrene and benzo(a)pyrene but cant
mineralize naphthalene.
The degradation potential of Mycobacterium PY146 in soil with and without
yeast extract was almost similar.
There is an effect of cell biomass on mineralization of phenanthrene and

pyrene.
There was negative effect of additional pyrene on radiolabled pyrene

mineralization.
Cloning of nidAB from Mycobacterium was successful but cant expressed in
E.coli in the present study. It might require its specific ferrodoxin and
reductase proteins for its expression.
There was a positive relation of RNA to DNA ratio of nidAB and
phenanthrene mineralization.
101
FutureProspects
Future prospects
PAH contaminated environments like soil and sediments can be exploited for
other bacterial strains capable of degrading PAHs.
These isolates can also be checked for their ability to degrade other organic
contaminants like pesticides, TNTs and PCBs etc.
These bacterial isolates can be applied in fields for bioremediation of PAHs
and other organic compounds.
Gene probes can be constructed to monitor PAH-degrading bacteria in
contaminated environments.
The expression of nidAB pyrene dioxygenase gene can be checked using other
competent E. coli cell lines and expression vectors.
The expression of monoxygenase gene could be checked and relate its
expression with the mineralization of PAHs.
Identification of metabolites from PAHs biotransformation to elucidate the
complete pathways by selected isolates.
102
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131
Appendix
Figure I a. Phylogenetic tree of bacterial isolates
132
Appendix
Percent Identity
1
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
99.8
99.6
94.0
99.8
99.7
99.8
99.8
93.9
98.9
94.1
99.8
93.6
78.1
99.3
99.2
82.0
94.5
94.8
78.8
78.5
78.8
93.0
92.3
92.2
78.0
84.9
76.6
B. cereus AB215098
98.9
93.5
98.6
98.6
98.6
98.6
92.8
96.6
93.0
99.5
93.6
77.9
99.3
99.2
81.5
94.0
94.3
78.8
78.5
78.8
93.0
92.3
92.2
77.2
84.9
76.7
B. cereus AF227848
94.1
99.7
99.6
99.7
99.7
94.0
97.6
94.1
99.7
93.6
78.7
99.3
99.2
81.7
94.0
94.3
78.9
78.6
78.8
92.7
92.0
91.8
77.9
85.0
76.7
AJ310098.seq
94.2
94.1
94.2
94.2
99.5
93.2
99.7
94.2
99.0
78.1
94.6
94.3
81.1
98.8
99.2
78.3
78.3
78.6
92.3
91.7
91.5
78.0
83.0
75.9
B. sonorensis AJ586363
99.9 100.0 100.0 94.1
96.8
94.2 100.0 93.8
78.1
99.3
99.2
81.7
94.0
94.3
78.8
78.5
78.8
93.0
92.3
92.2
78.4
84.9
76.7
B. cereus AY224379
94.1
96.8
94.2
93.7
78.0
99.3
99.2
81.7
94.0
94.3
78.7
78.4
78.7
92.9
92.3
92.1
78.3
84.8
76.7
B. cereus AY224383
100.0 94.1
96.8
94.2 100.0 93.8
78.1
99.3
99.2
81.7
94.0
94.3
78.8
78.5
78.8
93.0
92.3
92.2
78.4
84.9
76.7
B. cereus AY224385
94.1
96.8
94.2 100.0 93.8
78.1
99.3
99.2
81.7
94.0
94.3
78.8
78.5
78.8
93.0
92.3
92.2
78.4
84.9
76.7
AY224388.seq
91.9
99.8
94.0
99.3
77.9
94.5
94.2
81.0
98.7
99.1
78.3
78.3
78.6
92.3
91.8
91.6
78.2
83.0
75.6
B. licheniformis AY728013
92.3
98.2
94.3
81.0
99.4
99.3
81.2
93.7
94.0
81.8
81.8
83.2
93.0
93.1
92.4
80.3
86.5
79.4
10
B. Cereus DQ884352.seq
94.1
99.4
78.2
94.6
94.3
81.1
98.8
99.2
78.3
78.3
78.6
92.5
91.8
91.6
78.2
83.0
75.9
11
Bacillus sp.EF471917.seq
93.8
78.2
99.3
99.2
81.7
94.0
94.3
78.8
78.5
78.8
93.0
92.3
92.2
78.3
84.9
76.7
12
B. Cereus EU373359.seq
78.2
94.6
94.2
82.4
99.3
99.7
79.0
78.9
78.7
91.9
91.6
91.3
78.2
82.9
75.8
13
B. licheniformis EU373408.
82.1
82.0
98.4
81.1
81.3
98.6
99.9
96.8
77.4
77.3
76.7
94.1
75.9
84.8
14
Staph. haemolyticus EU652064
99.4
81.8
94.0
94.2
82.0
82.0
83.4
93.1
93.2
92.6
82.1
86.8
79.7
15
Fazal-1KWS4.seq
81.7
94.1
94.4
81.9
81.9
83.3
92.9
93.0
80.7
80.8
99.7
99.0
81.4
99.3
81.4
97.1
82.7
81.1
92.8
80.7
92.7
92.4
80.2
82.0
94.6
86.6
78.6
79.6
85.7
16
17
Fazal-2KWS2.seq
Fazal-3 10_1.seq
92.1
81.4
85.0
79.6
18
Fazal-4 DW3.seq
81.7
81.7
82.8
93.1
93.0
92.4
81.4
85.3
79.9
19
Fazal-5 10.seq
98.7
97.2
78.0
78.0
77.2
94.3
76.2
84.6
20
PSU26261.seq
96.9
77.5
77.4
76.8
94.3
75.9
84.8
21
P. stutzeri U26262
77.3
77.8
76.9
95.9
75.6
83.9
22
P. putida D37923.seq
97.8
97.8
76.8
82.4
76.3
23
Staph. aureus D83358
97.6
76.8
83.5
76.3
24
Staph. haemolyticus D83367
76.3
83.1
75.7
25
Staph. hyicus D83368
75.4
84.0
26
P. fluorescens Z76662
74.8
27
Strep. mutans X58303
28
E. coli X80725
0.2
0.3
1.0
6.2
6.7
6.0
0.2
1.4
0.3
6.0
0.3
1.4
0.3
6.1
0.1
0.2
1.4
0.3
6.0
0.0
0.1
0.2
1.4
0.3
6.0
0.0
0.1
0.0
6.4
7.6
6.2
0.5
6.1
6.2
6.1
6.1
1.1
3.4
2.4
7.2
3.3
3.3
3.3
3.3
8.6
6.2
7.4
6.1
0.3
6.0
6.1
6.0
6.0
0.2
8.2
0.2
0.5
0.3
6.0
0.0
0.1
0.0
0.0
6.2
1.8
6.2
6.7
6.7
6.7
0.9
6.6
6.6
6.6
6.6
0.7
6.0
0.6
6.6
26.0
26.3
25.2
26.0
26.1
26.2
26.1
26.1
26.3
22.0
25.8
26.0
26.0
0.7
0.7
0.7
5.6
0.7
0.7
0.7
0.7
5.7
0.6
5.6
0.7
5.6
20.5
11
12
13
14
15
16
17
10
99.9
99.9
99.9
0.8
0.8
0.8
6.0
0.8
0.8
0.8
0.8
6.1
0.7
6.0
0.8
6.1
20.6
0.6
18
20.3
5.7
20.7
6.3
20.5
6.3
21.2
1.2
20.5
6.3
20.5
6.3
20.5
6.3
20.5
6.3
21.4
1.3
21.1
6.6
21.2
1.2
20.5
6.3
20.1
0.7
0.9
22.0
20.3
6.3
20.5
6.2
21.9
19
5.4
5.8
5.8
0.7
5.8
5.8
5.8
5.8
0.8
6.2
0.7
5.8
0.3
21.5
6.0
5.8
21.7
0.2
20
25.0
25.0
24.8
25.7
25.0
25.1
25.0
25.0
25.8
21.0
25.8
25.0
24.8
1.4
20.7
20.8
0.7
21.5
21.2
21
25.5
25.5
25.3
25.7
25.5
25.6
25.5
25.5
25.8
21.0
25.8
25.5
24.9
0.1
20.7
20.8
0.5
21.5
21.2
1.3
22
25.1
25.1
25.0
25.3
25.1
25.2
25.1
25.1
25.4
19.2
25.4
25.1
25.2
3.2
18.9
19.0
2.9
19.8
19.7
2.8
3.2
23
7.4
7.4
7.7
8.1
7.4
7.5
7.4
7.4
8.2
7.5
8.0
7.4
8.6
27.0
7.3
7.6
21.6
7.7
7.3
26.1
26.9
27.2
24
8.2
8.2
8.5
8.8
8.2
8.3
8.2
8.2
8.7
7.3
8.7
8.2
9.0
27.3
7.2
7.4
22.2
7.8
7.5
26.2
27.1
26.6
2.2
8.3
8.3
8.7
9.1
8.3
8.4
8.3
8.3
9.0
8.1
9.0
8.3
9.4
28.2
7.9
8.2
23.0
8.4
8.1
27.5
28.0
27.8
2.3
2.5
25.6
26.7
25.6
25.5
25.0
25.1
25.0
25.0
25.3
22.7
25.4
25.2
25.2
5.6
20.3
20.4
4.7
21.3
21.1
5.4
5.4
3.6
27.4
27.4
28.1
14.7
14.7
14.6
17.1
14.7
14.8
14.7
14.7
17.2
13.2
17.2
14.7
17.3
26.9
12.7
13.0
23.2
15.0
14.6
26.5
26.9
27.4
17.9
16.5
17.0
27.0
27.9
27.8
27.8
29.0
27.8
27.8
27.8
27.8
29.5
24.0
29.0
27.8
29.2
16.8
23.5
23.6
15.5
23.6
23.3
17.1
16.9
17.9
28.4
28.4
29.4
17.2
28.3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
26
27
28
25
28
Figure I b. Percent identity of bacterial isolates with other known identified bacterial strains
133
Appendix
(Continued on next page)

134
Appendix
Figure I c. nidAB gene sequence cloned from Mycobacterium PY146 and containing
the EcoRI and HindIII restriction enzyme adaptor sequences
135
Appendix
Ab und ance
Scan 2744 (18.758 min): 0201002.D\ d ata .ms (-2724) (-)

115.0
7000000
6500000
144.0
6000000
5500000
5000000
4500000
4000000
3500000
3000000
2500000
2000000
1500000
1000000
89.0
63.0
500000
39.0
72.0
50.0
98.0
30
40
50
60
70
80
90
100
126.0135.0
110
120
130
140
m/ z-->
B
100
144
115
50
HO
89
63
39
50
72
98
30 40 50 60
(replib) 2-Naphthalenol
70
80
126
90 100 110 120 130 140
Figure II a. Mass spectra obtained by GC/MS of 2-Naphthalenol produced by Bacillus

cereus KWS2 grown with naphthalene (A) and from NIST mass spectral library (B).
136
Appendix
Ab und a nce
A
Sca n 2046 (15.025 min): 0201002.D\ d a ta .ms (-2025) (-)
91.0
200000
180000
160000
140000
120000
100000
80000
135.9
60000
40000
65.0
20000
39.0
51.0
77.0
107.1 117.9126.0
0
30
40
50
60
70
80
90
100
110
120
130
140
m/ z-->
B
91
100
O
OH
50
136
39 45 51
65
77
30 40 50 60 70
(replib)
Benzeneacetic acid
80
90 100 110 120 130 140
Figure II b. Mass spectra obtained by GC/MS of benzeneacetic acid produced by

Bacillus cereus KWS2 grown with naphthalene (A) and from NIST mass spectral
library (B).
137
Appendix
Ab und a nc e
Sc a n 1797 (13.693 min): 0201002.D \ d a ta .ms

105.0
8000
121.9
7500
7000
76.9
6500
6000
5500
5000
4500
4000
3500
3000
2500
43.0
51.0
2000
1500
59.9
1000
69.1
500
91.0
84.1
0
30
35
40
45
50
55
60
65
70
75
80
85
90
95 100105110115120125
m/ z-->
105
100
122
77
HO
51
50
39
45
94
65
30
40
50
60
70
(mainlib) Benzenecarboxylic acid
80
90
100
110
120
Figure II c. Mass spectra obtained by GC/MS of Benzenecarboxylic acid produced by

library (B).
138
Appendix
Ab und ance
Sca n 1153 (10.249 min): 0201002.D\ d ata .ms (-1141) (-)
130000
106.0
120000
110000
77.0
100000
90000
80000
70000
60000
51.0
50000
40000
30000
20000
39.0
10000
63.1
45.2
0
30
35
40
45
50
55
60
65
82.7 88.9
70
75
80
85
90
95.8
95 100 105
m/ z-->
B
106
77
100
51
50
30
39
74
43
30
40
50
(replib) Benzaldehyde
63
60
84 89
70
80
90
100
Figure II d. Mass spectra obtained by GC/MS of Bezaldehyde produced by Bacillus

139
Appendix
A
A b und a nc e
S c a n 4002 (25.487 m in): 0201002.D \ d a ta .m s (-4009) (-)
194.0
165.0
700000
650000
600000
550000
500000
450000
400000
350000
300000
250000
200000
150000
100000
82.1
139.0
50000
69.4
39.0
97.0
115.0
51.0
127.0
0
30
40
50
60
70
80
90
151.0
181.8
100 110 120 130 140 150 160 170 180 190
m/ z-->
B
194
100
165
50
37 49
69
82
OH
97
30
50
70
90
(mainlib)
9-Phenanthrenol
115 126 139 150

110
130
150
174
170
190
Figure II e. Mass spectra obtained by GC/MS of 9-phenanthrenol produced by

Bacillus cereus KWS2 grown in phenanthrene (A) and from NIST mass spectral
library (B).
140
Appendix
A b und a nc e
S c a n 3946 (25.187 m in): 0201002.D \ d a ta .ms (-3932) (-)

165.0
170000
160000
150000
140000
130000
120000
181.0
110000
212.0
100000
90000
80000
70000
60000
193.9
50000
152.0
40000
30000
20000
76.9
10000
115.0
63.0
49.9
138.9
101.9
0
30
40
50
60
70
80
90
100 110 120 130 140 150 160 170 180 190 200 210
m/ z-->
165
100
212
181
50
82
51 63
69
0
41
194
HO
OH
115
89
151
139
30
50
70
90 110 130 150 170
(mainlib)
9,10-Dihydro-9,10-dihydroxyphenanthrene
190
210
Figure II f. Mass spectra obtained by GC/MS of 9, 10-dihydro-9, 10dihydroxyphenanthrene produced by Bacillus cereus KWS2 grown in phenanthrene
(A) and from NIST mass spectral library (B).
141
Appendix
A b und a nc e
S c a n 2058 (15.089 min): 0201002.D \ d a ta .ms (-2025) (-)

91.0
400000
380000
360000
340000
320000
300000
280000
260000
240000
220000
200000
180000
160000
140000
135.9
120000
100000
80000
65.0
60000
40000
39.0
51.0
20000
77.0
98.8 106.9
0
30
40
50
60
70
80
90
100
110
118.0
120
128.2
130
m / z-->
B
91
100
O
OH
50
136
39 45 51
65
77
30 40 50 60 70
(replib)
Benzeneacetic acid
80
90 100 110 120 130 140
Figure II g. Mass spectra obtained by GC/MS of benzeneacetic acid produced by

library (B).
142
Appendix
Ab und a nce
Sca n 2856 (19.357 min): 0301003.D \ d a ta .ms (-2816) (-)

107.0
1100000
1000000
900000
800000
700000
600000
500000
400000
151.9
300000
77.0
200000
100000
51.0
39.0
63.0
89.0 98.0
0
30
40
50
60
70
80
90
129.0
116.9
100
110
120
141.2
130
140
150
m/ z-->
107
100
O
OH
50
152
77
0
31 39
51
OH
63
86 95
121
134
30 40 50 60 70 80 90 100 110 120 130 140 150

(mainlib)
Benzeneacetic acid, 4-hydroxy
Figure II h. Mass spectra obtained by GC/MS of 4-hydroxy benzeneacetic acid

produced by Bacillus cereus KWS2 grown in phenanthrene (A) and from NIST mass
143
Appendix
A
A b und a nc e
S c a n 1862 (14.041 min): 2301024.D \ d a ta .ms (-1871) (-)
105.0
95000
90000
85000
122.0
80000
75000
70000
65000
60000
77.0
55000
50000
45000
40000
35000
30000
51.0
25000
20000
15000
10000
39.0
5000
60.0
94.1
85.0
69.0
115.0
0
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95 100105110115120
m/ z-->
105
100
HO
122
77
50
51
39 45
74
65
94
0
30
40
50
60
70
(replib) Benzenecarboxylic acid
80
90
100
110
120
Figure II i. Mass spectra obtained by GC/MS of benzenecarboxylic acid produced by

Bacillus cereus KWS2 grown in anthracene (A) and from NIST mass spectral library
(B).
144
Appendix
Ab und a nce
A
Sca n 2088 (15.250 min): 2301024.D\ d a ta .ms (-2075) (-)
91.0
220000
200000
180000
160000
140000
120000
100000
136.0
80000
60000
101.0
40000
65.0
39.0
20000
116.0
75.0
51.0
128.1
0
30
40
50
60
70
80
90
100
110
120
130
m/ z-->
91
100
O
OH
50
136
65
39 46 51
56
72 77
30 40 50 60 70
(replib)
Benzeneacetic acid
80
101
90
117
100 110 120 130 140
Figure II j. Mass spectra obtained by GC/MS of benzeneacetic acid produced by

Bacillus cereus KWS2 grown in anthracene (A) and from NIST mass spectral library
(B).
145
Appendix
A b und a nc e
S c a n 2856 (19.357 min): 2301024.D \ d a ta .ms (-2826) (-)

107.0
800000
750000
700000
650000
600000
550000
500000
450000
400000
350000
300000
152.0
250000
200000
77.0
150000
100000
51.1
50000
39.1
63.0
89.0
0
30
40
50
60
70
80
90
123.1
100
110
120
141.9
130
140
150
m/ z-->
107
100
O
OH
50
152
77
0
31 39
51
63
OH
86 95
121
134
30 40 50 60 70 80 90 100 110 120 130 140 150

(mainlib)
Figure II k. Mass spectra obtained by GC/MS of 4-hydroxy benzeneacetic acid

produced by Bacillus cereus KWS2 grown in anthracene (A) and from NIST mass
146
Appendix
A b und a nc e
S c a n 2740 (18.737 m in): 0601006.D \ d a ta .ms (-2721) (-)

115.0
3200000
144.0
3000000
2800000
2600000
2400000
2200000
2000000
1800000
1600000
1400000
1200000
1000000
800000
600000
89.0
400000
63.0
200000
39.0
74.0
50.0
98.0
126.0134.8
0
30
40
50
60
70
80
90
100
110
120
130
140
m / z-->
B
100
144
115
50
HO
89
63
39
50
72
98
0
30 40 50 60
(replib) 2-Naphthalenol
70
80
126
90 100 110 120 130 140 150
Figure III a. Mass spectra obtained by GC/MS of 2-naphthalenol produced by

library (B).
147
Appendix
A b und a nc e
S c a n 2045 (15.020 min): 0601006.D \ d a ta .ms (-2021) (-)
91.0
180000
170000
160000
150000
140000
130000
120000
110000
100000
90000
80000
70000
135.9
60000
50000
40000
65.0
30000
20000
39.0
51.0
10000
76.9
99.8 108.0 118.0 127.9
0
30
40
50
60
70
80
90
100
110
120
130
140
m/ z-->
B
91
100
O
OH
136
50
65
39
44
51
77
105
0
30 40 50 60 70
(mainlib) Benzeneacetic acid
80
90
118
100 110 120 130 140
Figure III b. Mass spectra obtained by GC/MS of benzeneacetic acid produced by

library (B).
148
Appendix
Ab und a nce
A
Sca n 1151 (10.238 min): 0601006.D \ d a ta .ms (-1140) (-)
106.0
77.0
20000
18000
16000
14000
12000
10000
51.0
8000
6000
4000
2000
39.0
62.9
45.0
0
30
35
40
45
50
55
60
88.0
70.9
65
70
75
80
85
90
95
100 105
m/ z-->
B
106
77
100
51
50
30
39
74
43
30
40
50
(replib) Benzaldehyde
63
60
84 89
70
80
90
100
Figure III c. Mass spectra obtained by GC/MS of Bezaldehyde produced by Bacillus

149
Appendix
A
Ab und a nc e
S c a n 4002 (25.487 min): 0401004.D \ d a ta .ms (-4012) (-)

165.0
194.0
1050000
1000000
950000
900000
850000
800000
750000
700000
650000
600000
550000
500000
450000
400000
350000
300000
250000
200000
150000
82.2
139.0
100000
69.4
50000
97.0
39.0 51.0
0
30
40
50
60
70
80
90
115.0
127.0
150.9
179.9
100 110 120 130 140 150 160 170 180 190
m/ z-->
165
100
194
50
82
39 50
69
OH
87 97
0
30
50
70
(replib) 9-Phenanthrenol
90
115 126
110
139
130
150
150
174
170
190
Figure III d. Mass spectra obtained by GC/MS of 9-phenanthrenol produced by

library (B).
150
Appendix
A
A b und a nc e
S c a n 3947 (25.193 min): 0401004.D \ d a ta .m s (-3931) (-)
165.0
200000
190000
180000
170000
160000
150000
140000
181.0
130000
120000
212.0
110000
100000
90000
80000
70000
60000
151.9
50000
40000
30000
77.0
20000
194.9
115.0
138.9
63.0
10000
50.0
0
101.8
36.9
30
40
50
60
70
80
90
100 110 120 130 140 150 160 170 180 190 200 210
m/ z-->
B
165
100
212
181
50
82
51 63
69
0
41
194
HO
89
OH
115
151
139
30
50
70
90 110 130 150 170
(mainlib) 9,10-Dihydro-9,10-dihydroxyphenanthrene
190
210
Figure III f. Mass spectra obtained by GC/MS of 9,10-dihydro-9,10dihydroxyphenanthrene produced by Bacillus cereus KWS4 grown in phenanthrene
(A) and from NIST mass spectral library (B).
151
Appendix
A
Ab und a nc e
Sc a n 2065 (15.127 min): 0401004.D \ d a ta .ms (-2026) (-)
91.0
600000
550000
500000
450000
400000
350000
300000
250000
135.9
200000
150000
65.0
100000
39.0
50000
51.0
77.0
107.0
0
30
40
50
60
70
80
90
100
110
117.8 126.8
120
130
m/ z-->
B
91
100
O
OH
50
136
39 45 51
65
77
30 40 50 60 70
(replib) Benzeneacetic acid
80
90
100 110 120 130
Figure III g. Mass spectra obtained by GC/MS of benzeneacetic acid produced by

library (B).
152
Appendix
A
Ab und a nc e
Sc a n 2841 (19.277 min): 0501005.D \ d a ta .ms (-2810) (-)

107.0
170000
160000
150000
140000
130000
120000
110000
100000
90000
80000
70000
151.9
60000
50000
77.0
40000
30000
63.0
51.0
20000
86.9
125.9
39.0
10000
97.9
0
30
40
50
60
70
80
90
100
134.9
116.0
110
120
130
140
150
m/ z-->
B
107
100
O
OH152
50
77
39
0
30
51
HO
63
90
124 134
30 40 50 60 70 80 90 100 110 120 130 140 150

(replib)
Figure III h. Mass spectra obtained by GC/MS of 3-hydroxy benzeneacetic acid

produced by Bacillus cereus KWS4 grown in phenanthrene (A) and from NIST mass
153
Appendix
Ab und a nc e
Sca n 2218 (15.945 min): 0501005.D \ d a ta .ms (-2210) (-)

92.0
119.9
60000
55000
50000
45000
40000
137.9
35000
30000
25000
20000
64.0
43.0
15000
10000
53.0
72.0
5000
0
80.8
105.1
35.1
30
40
50
60
70
80
90
100
110
127.9
120
130
140
m/ z-->
B
92
100
120
O
OH
138
50
OH
64
39
45
53
74 81
109
0
30 40 50 60 70 80
(replib) Benzoic acid, 2-hydroxy-
90
100 110 120 130 140
Figure III i. Mass spectra obtained by GC/MS of 2-hydroxy benzoic acid produced by
library (B).
154
Appendix
Abundance
Scan 1857 (17.014 min): 2001005.D\ data.ms

104.0
600000
550000
500000
450000
76.0
400000
350000
300000
250000
200000
50.0
150000
148.0
100000
50000
38.0
61.0
85.0
0
30
40
50
60
70
80
116.0
94.8
90
100
110
120
128.9 138.0
130
140
m/ z-->
B
104
100
76
OH
50
OH
50
O
31
38
61
148
85
30 40 50 60 70 80 90 100 110 120 130 140 150

(rep
lib
) 1,2-Benzened icarb oxylic acid
Figure IV a. Mass spectra obtained by GC/MS of 1, 2-benzenecarboxylic acid

produced by resting cells of Mycobacterium PY146 with phenanthrene (A) and from
155
Appendix
Abundance
A
Scan 1865 (17.057 min): 2201007.D\ data.ms (-1849) (-)
104.0
30000
28000
26000
24000
22000
76.0
20000
18000
16000
14000
12000
10000
8000
148.0
50.1
6000
4000
37.2
2000
60.8
30
40
50
60
116.7 126.9
93.0
0
70
80
90
100
110
120
130
140
m/ z-->
104
100
76
OH
50
OH
50
O
31
38
61
148
85
30 40 50 60 70 80 90 100 110 120 130 140 150

(rep lib ) 1,2-Benzened icarb oxylic a cid
Figure IV b. Mass spectra obtained by GC/MS of 1,2-benzenecarboxylic acid

produced by resting cells of Mycobacterium PY146 with anthracene (A) and from
156
Appendix
Abundance
Scan 3359 (25.048 m

in): 2101006.D\ data.m
s
208.0
160000
150000
140000
180.0
130000
120000
110000
100000
152.0
90000
80000
70000
60000
50000
40000
76.0
30000
20000
50.0
10000
0
126.0
63.0
90.0
111.0
30
40
50
60
70
80
90
194.0
165.0
139.0
36.8
100 110 120 130 140 150 160 170 180 190 200
m
/ z-->
208
100
180
152
50
76
O
39
50 63
90
99
30
50
70
90
110
(replib)
9,10-Anthracenedione
126
130
163
150
170
190
210
Figure IV c. Mass spectra obtained by GC/MS of 1,2-benzenecarboxylic acid

produced by resting cells of Mycobacterium PY146 with anthracene (A) and from
157
Appendix
Abundance
Scan 1861 (17.035 min): 2401009.D\ data.ms (-1849) (-)

104.0
40000
35000
30000
76.0
25000
20000
15000
10000
50.0
148.0
5000
38.1
61.1
87.1
113.0
0
30
40
50
60
70
80
90
100
110
124.9 133.7
120
130
140
m/ z-->
104
100
76
OH
50
OH
50
O
31
38
61
148
85
30 40 50 60 70 80 90 100 110 120 130 140 150

(replib)
1,2-Benzenedicarb oxylic acid
Figure IV d. Mass spectra obtained by GC/MS of 1,2-benzenecarboxylic acid

produced by resting cells of Mycobacterium PY146 with pyrene (A) and from NIST
158
Appendix
Abundance
Scan 4817 (32.846 m

in): 2301008.D\ data.m
s
218.0
24000
23000
22000
21000
20000
19000
18000
17000
16000
15000
14000
13000
12000
11000
189.0
10000
9000
8000
7000
6000
5000
4000
3000
43.0
2000
1000
57.1
72.9
94.1
111.0
130.0
147.0
163.1
203.0
0
30
40
50
60
70
80
90
100 110 120 130 140 150 160 170 180 190 200 210 220
m
/ z-->
218
100
OH
189
50
94
81
109
163
0
30
50
70
90
(rep
lib
)
1-Hyd
roxypyrene
110
130
150
170
190
210
Figure IV e. Mass spectra obtained by GC/MS of 1-hydroxypyrene produced by

resting cells of Mycobacterium PY146 with pyrene (A) and from NIST mass spectral
library (B).
159
Appendix
Ab und a nc e
2600000
Sc a n 2241 (16.068 min): 1701018.D \ d a ta .ms (-2216) (-)

104.0
2400000
2200000
76.0
2000000
1800000
1600000
1400000
1200000
1000000
50.1
800000
600000
148.0
400000
38.1
200000
61.0
85.0
0
30
40
50
60
70
80
94.9
90
118.9 129.0 139.0
100
110
120
130
140
m/ z-->
104
100
76
OH
50
OH
50
O
31
38
61
148
85
30 40 50 60 70 80 90 100 110 120 130 140 150

(replib) 1,2-Benzenedicarboxylic acid
Figure V a. Mass spectra obtained by GC/MS of 1,2-benzenedicarboxylic acid

produced by Mycobacterium PY146 grown in phenanthrene (A) and from NIST mass
160
Appendix
Ab und a nce
A
450000
Sca n 2068 (15.142 min): 1901020.D\ d a ta .ms (-2034) (-)

91.0
400000
350000
300000
250000
200000
136.0
150000
100000
65.0
50000
39.1
51.0
77.0
98.9 107.0
0
30
40
50
60
70
80
90
100
110
117.9
120
130
m/ z-->
91
100
O
OH
50
136
39 45 51
65
30 40 50 60 70
(replib)
Benzeneacetic acid
77
80
90
100 110 120 130
Figure V b. Mass spectra obtained by GC/MS of benzeneacetic acid produced by

Mycobacterium PY146 grown in phenanthrene (A) and from NIST mass spectral
library (B).
161
Appendix
A b und a nc e
Sc a n 3709 (23.920 min): 3001031.D \ d a ta .ms (-3691) (-)

180.0
90000
208.0
85000
80000
75000
70000
65000
152.0
60000
55000
50000
45000
40000
35000
30000
76.0
25000
20000
15000
50.0
10000
126.0
63.0
5000
0
97.9
111.0
37.0
30
40
50
60
70
80
90
194.9
100 110 120 130 140 150 160 170 180 190 200
m/ z-->
152
100
180
208
76
50
50
63
0
39
126
90 102
30
50
70
90 110
(replib) 9,10-Anthracenedione
130
150
170
190
210
Figure V c. Mass spectra obtained by GC/MS of 9,10-anthracenedione produced by

Mycobacterium PY146 grown in anthracene (A) and from NIST mass spectral library
(B).
162
Appendix
Ab und a nce
A
Sca n 1834 (13.891 min): 3101032.D \ d a ta .ms (-1788) (-)
105.0
50000
45000
122.0
40000
77.0
35000
30000
25000
20000
51.0
15000
10000
43.1
87.0
60.0
5000
94.0
70.0
115.1
0
25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100105110115120
m/ z-->
105
100
HO
122
77
50
51
39 45
65
74
94
0
30
40
50
60
70
(replib)
Benzenecarboxylic
acid
80
90
100
110
120
Figure V d. Mass spectra obtained by GC/MS of benzenecarboxylic acid produced by

(B).
163
Appendix
Ab und a nc e
Sc a n 2080 (15.207 min): 3101032.D \ d a ta .ms (-2031) (-)

91.0
700000
650000
600000
550000
500000
450000
400000
350000
300000
136.0
250000
200000
150000
65.0
100000
39.1
50000
51.0
77.0
99.0 107.0
0
30
40
50
60
70
80
90
100
110
118.0 127.8
120
130
m/ z-->
91
100
O
OH
50
136
39 45 51
65
30 40 50 60 70
(replib) Benzeneacetic acid
77
80
90
100 110 120 130
Figure V e. Mass spectra obtained by GC/MS of benzeneacetic acid produced by

(B).
164
Appendix
A b und a nc e
S c a n 2234 (16.030 min): 3101032.D \ d a ta .ms (-2218) (-)

104.0
200000
190000
180000
170000
160000
150000
76.0
140000
130000
120000
110000
100000
90000
80000
70000
50.0
60000
50000
40000
147.9
30000
20000
38.0
10000
61.0
0
20
30
40
50
60
112.9122.1
92.1
70
80
90
100
110
120
136.0
130
140
150
m/ z-->
B
104
100
76
OH
50
OH
50
O
31
38
61
148
85
30 40 50 60 70 80 90 100 110 120 130 140 150

Figure V f. Mass spectra obtained by GC/MS of 1,2-benzenedicarboxylic acid

produced by Mycobacterium PY146 grown in anthracene (A) and from NIST mass
165
Appendix
Ab und a nc e
Sc a n 1614 (12.956 min): 0501005.D \ d a ta .ms (-1598) (-)

105.0
122.0
55000
50000
45000
77.0
40000
35000
30000
25000
20000
51.1
15000
87.0
10000
60.0
5000
94.0
69.0
113.0
0
45
50
55
60
65
70
75
80
85
90
95
100 105 110 115 120
m/ z-->
105
100
122
77
HO
51
50
65
74
90
0
50
60
70
80
(mainlib) Benzenecarboxylic acid
90
94
100
110
120
Figure V g. Mass spectra obtained by GC/MS of benzenecarboxylic acid produced by

Mycobacterium PY146 grown in pyrene (A) and from NIST mass spectral library (B).
166
Appendix
Ab und a nce
Sc a n 1887 (14.373 min): 0501005.D \ d a ta .ms

91.1
900000
800000
700000
600000
500000
400000
136.0
300000
200000
65.1
79.1
100000
51.1
58.0
98.1
72.1
122.0
107.1 115.0
129.1
40 45 50 55 60 65 70 75 80 85 90 95 100105110115120125130135
m/ z-->
B
91
100
O
OH136
50
65
51
63
89
77
105
0
50
60
70
80
(mainlib)
Benzeneacetic
acid
90
100
110
118
120
130
140
Figure V h. Mass spectra obtained by GC/MS of benzeneacetic acid produced by

Mycobacterium PY146 grown in pyrene (A) and from NIST mass spectral library (B).
167
Appendix
Ab und a nc e
Sc a n 2014 (15.032 min): 0501005.D \ d a ta .ms (-1993) (-)

104.0
1300000
1200000
1100000
1000000
76.0
900000
800000
700000
600000
500000
50.1
400000
300000
148.0
200000
100000
61.0
85.0
0
40
50
60
70
80
118.9127.1 136.8
93.0
90
100
110
120
130
140
m/ z-->
104
100
76
OH
50
50
OH
148
O
61 66
85
50 60 70 80 90 100 110 120

130 140 150
Figure V i. Mass spectra obtained by GC/MS of 1,2-benzenedicarboxylic acid

produced by Mycobacterium PY146 grown in pyrene (A) and from NIST mass
168

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Biodegradation of Polycyclic Aromatic Hydrocarbons

Biodegradation of Polycyclic Aromatic Hydrocarbons

A thesis submitted in partial fulfillment of the requirements

Internal Examiner: ____________________

External Examiner: _____________________

External Examiner: _____________________

Dated: May 27, 2011

Chapter 2: Review of Literature

Chapter 3: Materials and Methods

3.2 SELECTION OF PAH TRANSFORMING BACTERIA

3.2.1 Culture condition and media

3.2.2 Screening of PAHs degrading bacteria

3.2.3 Identification of selected bacterial isolates

3.2.3.1 Morphological and biochemical characterization

3.2.3.2 Molecular characterization based on 16S rRNA

cereus KWS2 and P. stutzeri 10-1

KWS2 and B. cereus KWS4

3.3.4 Biodegradation of PAHs in Soil

3.3.5 Analytical methods

3.3.5.1 Viable Cell Counts

3.3.5.2 HPLC Analysis

3.3.5.3 Extraction and Analysis of PAHs and their

3.4 MINERALIZATION STUDIES OF PAHs

3.4.1 14C-PAH mineralization assay

3.4.2 14C-Pyrene and phenanthrene mineralization using

different cells densities

FROM MYCOBACTERIUM PY146 into E. coli

3.5.2 Amplification of nidAB

3.5.3 Cloning of nidAB gene in E. coli

3.5.3.1 Cloning of nidAB into pCR 2.1 TOPO vector

3.5.3.2 Transformation of E. coli with recombinant pCR

2.1 TOPO vector

from transformed E. coli cells

3.5.3.5 Addition of restriction sites on the ends of nidAB

3.5.3.6.2 Gel Purification of fragments

3.5.3.6.3 Ligation of EcoRInidAB HindIII and

3.5.3.6.6 Restriction enzyme analysis of

recombinant pkk223-3 vector

plasmid contained EcoRInidAB HindIII

3.5.5.1 Linearizing the nidAB DNA template

3.5.5.2 Synthesis of large quantities of RNA

3.5.5.3 Isolation of RNA transcript of nidAB

3.5.6 Quantification of DNA and RNA copy number of nidAB

4.1.1 Identification of selected bacterial isolates

4.1.1.1 Morphological and biochemical characterization

4.1.1.2 Molecular characterization based on 16S rRNA gene

4.2 BIODEGRADATION STUDIES OF PAHS

cereus KWS2 and P. stutzeri 10-1

KWS2 and KWS4

4.2.4 Biodegradation of PAHs in soil

4.3 MINERALIZATION STUDIES OF PAHs

C-PAH mineralization by Bacillus cereus KWS2 and

C-Pyrene and phenanthrene mineralization using

different cells densities

FROM MYCOBACTERIUM PY146

4.4.2 Biotransformation of PAHs by E. coli with cloned nidAB

real-time PCR with PAH mineralization in Mycobacterium

Basal salt medium

Denaturing gradient gel electrophoresis

Dense nonaqueous phase liquids

Gas chromatography/mass spectrometry

Genetically engineered microorganisms

High molecular weight