Degradation pathway of pyrene in
Novosphingobium pentaromativorans
US6-1: Potential for bioremediation of
PAHs contaminated environment
Korea Ocean Research & Development Institute (KORDI)
Yuanrong Luo, Kaekyoung Kwon, Seunghyuk Lee，Sangjin Kim,
Sungho Yoon, Youngho Chung
Feb. 26, 2010
Outline
 Organic pollutants and marine pollution
 Overview of microbial degradation of PAHs
 Novosphingobium pentaromativorans US6-1
 Metabolic pathway using proteomic and genomic
approaches
 Conclusions
 Organic pollutants
 Persistent Organic Pollutants (POPs)
 Polycyclic Aromatic Hydrocarbons (PAHs)
 High Molecular Weight PAHs (HMW PAHs)
 Pyrene
 Benzo(a)pyrene (BaP)
High molecular weight PAHs (HMW PAHs)
Loss rates (half life)
Fluoranthene
Chrysene
Pyrene
Benzo[a]pyrene
Benzo[a]anthracene
Dibenz[a,h]anthracene
•
2 ring PAH - 3 week
•
4 ring PAH - 1 year
•
5 ring PAH - 6 years
PAHs in the marine environment
Anthropogenic inputs of PAHs from oil spills, ship traffic,
urban runoff, wastewater and industrial discharge, as well as
atmospheric fallout of vehicle exhaust and industrial stack
emission have caused significant accumulation of PAHs in
the marine environment.
What is Bioremediation?
Bioremediation
Biology
“Remediate”= To solve a problem
Bio-Remediate= to use biological organisms to solve
an environmental problem
 Bioremediation is a treatment process that uses
microorganisms (bacteria, yeast, or fungi) to break
down, or degrade, hazardous substances into less
toxic or nontoxic substances.
2.
1.
Microbe
3.
CO2+H2O
CO2+H2O
Oil
CO2+H2O
Although the metabolism of HMW PAHs has been studied
for over 30 years, less is known about the HMW PAH
metabolic pathways, genes, and enzymes than about those
for LMW aromatic hydrocarbons.
OUR INTERESTS:
To find microorganisms capable of breaking down
the PAHs, to determine degradation characteristics, to
elucidate HMW PAH biodegradative pathways and
implications in bioremediation.
Selection & Identification of potent PAHs degrading bacteria:
Novosphingobium pentaromativorans US6-1
Chrysene
Relative intensity
100
80
60
PAH
US6-1+PAH
US6-1
40
Rhodospirillum rubrum ATCC 11170 T (D30778)
20
Acetobacter aceti DSM 3508 T (X94066)
Sandaracinobacter sibricus RB16-17T (Y10678)
0
Sphingomonas paucimobilis ATCC 10829 T (D16144)
1000
0
20
40
60
Zymomonas mobilis ZM4T (AF086792)
Sphingobium yanoikuyae IFO 151027 T (X85023)
615
Novosphingobium pentaromativorans US6-1 KCTC 10454 T (AF502400)
Benz[a]Pyrene
Relative intensity
700
456
Novosphingobium subarcticum KF-3T (X94104)
666
Novosphingobium rosa IFO 15208T (D13945)
863
600
Novosphingobium stygium IFO 16085T (AB25013)
830
500
748
Novosphingobium tardaugens ARI-1T (AB070237)
547
Novosphingobium hassiacum W-51T (AJ416411)
437
400
704
300
613
995
PAH
US6-1+PAH
US6-1
200
100
996
Time (Min)
Porphyrobacter neustonensis DSM 9434T (AB033327)
Sphingopyxis terrae IFO 15098T (D13727)
0
20
Novosphingobium aromaticiborans IFO 16084 T (AB025012)
Erythrobacter longus DSM 6997 T (AF465835)
0.03
0
Novosphingobium capsulata GIFU 11526 T (D16147)
Novosphingobium subterraneum IFO 16086T (AB025014)
40
60
969
846
Sphingopyxis macrogoltabidus IFO 15033T (D13723)
Sphingopyxis alaskensis AF01T (AF378796)
Novosphingobium pentaromativorans US6-1

Isolated from muddy sediment of Ulsan
Bay, Republic of Korea in 2004.

Gram-negative, yellow-pigmented,
halophilic

It has broad substrates that can degrade
HMW-PAHs of two to five rings,
especially with the ability of degrading
Benzo(a)pyrene at high efficiency.
Novosphingobium pentaromativorans US6-1
Reaction
Gram form
-
Oxidase
-
Catalase
+
Cell shape
Colony color
Rod
2.5
OD at A660nm
Characteristics
Yellow
1.5
1.0
0.5
0.0
Growth condition
Temperature (°C)
30 (30-37)
pH
6.2 (5.5-8)
10
+
-glucosidase (Esculin)
+
-galactosidase
-
Indol production
-
Urease
-
Actdification from glucose
-
Arginine dihydrogenase
-
+
Maltose
+
Phenyl-acetate
+
Isoprenoid quinone
DNA G+C content (mol %)
Main fatty acid
Q-10
61.1
18:1,
14:0-OH
50
1.5
1.0
0.5
0.0
4
5
6
7
8
9
10
7
8
pH
2.5
OD at A660 nm
Glucose
40
2.0
3
Assimilation of :
30
2.5
2.4(2-3)
Reduction of nitrate to nitrogen
20
Temperature (oC)
OD at A660nm
NaCl requirements(%)
2.0
2.0
1.5
1.0
0.5
0.0
0
1
2
3
4
5
NaCl conc. (% , w/v)
6
Degradation Characteristics: Optimal condition of PAHs
biodegradation by strain US6-1
Conc. of BaP (ppm)
12
10
o
8
10 C
o
20 C
o
25 C
o
30 C
o
35 C
o
40 C
6
4
2
0
0
2
4
6
8
Time (days)




Medium : MM2 inorganic nutrients
COndition : BaP 10 ppm, 8 days
Analysis : GC/FID
pH control : Use Biological buffer
Results ;
45.0
 Temperature should be maintained
higher than 20 oC
 Optimal temperature = 35 oC
 Should maintain pH 4.0~9.0
 Optimal pH = 5.0~8.0
40.0
BaP Degraded(%)
Purpose ; Optimization of
biodegradation by strain US6-1 for
the application on bioreactor
Methods ;
35.0
30.0
25.0
20.0
15.0
10.0
5.0
0.0
4
5
6
7
pH
8
9
H
A
A
m
)
10
( p
p
8
s
6
4
r o
t e i n
( u
g
/ m
l )
2
0
C
0 .1 2
0 .1 0
0 .0 8
C o n t r o l- n o P A H
M ix e d P A H s
P y re n e
B e n z [a ]a n th ra c e n e
C h ry s e n e
B e n z [ b ] f lu o r a n t h e n e
B e n z o [a ]p y re n e
0 .0 6
0 .0 4
0 .0 2
0 .0 0
0
2
4
T im e (d a y s )
6
8
o
n
t r a t i o
6
Methods ;
Media : MM2 inorganic medium
Condition : BaP 10 ppm, till to 8 days
Analysis : GC/FID
c e n
8
n
10
o
12
0
f
P
2
B
P
Purpose ; Understanding the
biodegradation of PAHs by
strain US6-1
A
H
4
C
C
o
n
c e n
t r a t i o
n
o
f
P
Degradation Characteristics: Substrate utilization process
Results ;
 The order of biodegradation
was different between as a
single substrate and mixture of
PAHs compounds
 -HPCD was used as growth
promoting substrate
Degradation Characteristics: Effect of -HPCD on the degradation of PAHs
Purpose ; Enhancing the
biodegradation rate of PAHs by
strain US6-1
Methods ;
Concentration of PAHs (ppm)
100
80
 Media : MM2 inorganic medium
 Condition : BaP 10 ppm, 1 week
interval
 Analysis : GC/FID
 Option : Add 10% -HPCD
60
40
20
 Results ;
0
Phe
Pyr
Chr
BaA
BbF
Compounds
BaP
DBA
 The degradation of PAHs larger
than 4 ring was greatly enhanced
by the addition of -HPCD
Degradation Characteristics: Effect of concentration of -HPCD on the
degradation of PAHs
Purpose ; Enhancing the
biodegradation rate of PAHs by
strain US6-1
Methods ;
Benzo[a]pyrene (ppm)
10
8
 Media : MM2 inorganic medium
 Condition : BaP 10 ppm, 1 week
interval
 Analysis : GC/FID
 Option : 0.001% yeast extract or
1% or 10% -HPCD
Sterilized Control
W/O -HPCD
-HPCD (10%)
YE + -HPCD (10%)
YE only
YE + -HPCD (1%)
6
4
2
 Results ;
0
0
7
14
21
28
Time (days)
35
42
49
 The biodegradation rate was
corresponded with the conc. of HPCD
 The addition of yeast extracts inhibit
BaP biodegradation regardless of HPCD
 w/o:without
Genomic and proteomic
approaches in the elucidation
of pyrene biodegradative
pathway
Schematic flow diagram
Genome
Proteome
Metabolome
SDS-PAGE, 2D-E,
LC-MS, NMR,
LC-MS/MS
GC-MS
Bioinformatic analysis
Bioinformatic analysis
Genome sequencing
Characterize the functional
genes involved in
PAH degradation
Identify proteins that
induced by PAHs
Compare, Cluster
& Integrate
Elucidate the metabolic pathways
in Strain US6-1
Analyze the metabolic
intermediates
Genomic analysis
 Sequencing by using 454 pyrosequencing system
 Approx. 5.3 Mb of genome coded 5197 genes
 Approx. 25%(1300 ORFs) are likely directly associated with
catabolism or transport of PAH compounds, genes that
encode enzymes associated with the degradation of
fluorene, anthracene, benzoate, biphenyl, naphthalene, and
citrate cycle (TCA cycle) are predicted to be distributed
among obtained sequences. 25-30% of the genes are
related with amino acids metabolism, the rest proteins are
involved in ATP metabolism or have no obvious homology to
known genes.
Genomic analysis
Genes involved in the upper PAH degradation:
pathway of
 Ring-hydroxylation step
Following reactions:
aromatic diol to hydroxy
carboxyaromatic
Monooxygenation reaction:
PAH→PAH-epoxide→PAH-diol (Fig 1)
(5)
(1)
(2)
(1) Monooxygenase, (2)Epoxide hydrolase
(6)
Dioxygenation reaction
PAH→PAH-dihydrodiol→PAH-diol→PAHaldehyde (Fig 2)
(3)
＋
O2
(4)
(3)Ring hydroxylating dioxygenase, (4)Dihydrodiol hydrogenase
〔
〕
A
B
Genes involved in the lower pathway of PAH degradation
Ring cleavage: extradiol
C
(7)
1. Salicylate pathway: through catechol (catecol
2,3-dioxygenase, meta-cleavage) or gensitate
(gentisate 1,2-dioxygenase, para-cleavage) and
then mineralized to CO2 via TCA cycle.
D
(8)
(9)
E
F
(10)
C:Salicylaldehyde, D: Salicylate, E: Catechol, F:
Gentisate, G:2-Hydroxy-muconate semialdehyde,
H:Maleypyruvate, I: Pyruvate
(7):Salicyaldehyde dehydrogenase, (8):Salicylate 1hydroxylase, (9):Salicylate 5-hydroxylase, (10):
Catechol 2,3-dioxygenase, (11): Gentisate 1,2dioxygenase
(11)
H
G
TCA
cycle
I
2.o-phthalate pathway: through protocatechuate and then
mineralized into CO2 via TCA cycle.
J
L
K
(13)
(12)
(14)
J: 4-Hydroxybenzoate K:4,5-Dihydroxyphthalate
L:Vanillate M:3,4-Dihydrobenzoate
(Procatechuate) N:2-Hydroxy-4carboxymuconate semialdehyde(3)-CHMS, O:
Pyruvate
(12): 4-hydroxybenzoate monoxygenase;
(13):4,5-Dihydroxyphthalate decarboxylase, (14):
Vanillate monoxygenase; (15): Protocatechuate
4,5-dioxygenase (PCD45)
M
(15)
N
TCA cycle
O
Proteomic approaches
Benzo(a)
pyren
11
Pyrene induced, uninduced
10
9
8
7
1D: SDS-PAGE
6
5
4
2D-E: two dimensional electrophoresis
3
2
1
Pyren
Phenant ZB
hren
(pyren) 3hrs
Pyren
24hrs
MA
MM2
Pyren
Pyrene,
Benzo(a)pyreneinduced
Pyrene,
Benzo(a)pyreneuninduced
2
1
4
13
3
5
6
14
7
17
65
66
68
8
69
16
20
31
51
52
100
125
124
126
141
107
111
127
114
137
177
119
138
136
151
150
152
149
148
156
161
173
122
120
147
170
160
176
121
118
140
135
163
172
95
94
117
139
146
159
93
92
145
144
143
90
134
133
132
142
49
115
112
130
131
63
91
87
110
48
46
89
88
113
47
45
84
116
129
44
62
86
85
109
28
72
43
42
60
59
82
81
76
128
105
101
99
40
83
108
104
41
64
61
77
96
98
37
78
106
27
25
57
80
12
24
39
58
103
102
23
38
36
35
54
79
73
22
56
75
74
26
21
67
34
55
53
97
33
71
32
30
123
15
19
18
70
50
29
11
10
9
164
162
171
169
167
153
157
165
154
168
166
180
155
158
182
181
232
178
186
179
174
183
231
201
184
195
175
199
197
196
204
203
185
206
221
215
209
208
207
218
222
220
227
224
228
213
214
225
226
223
193
191
192
219
217
216
212
189
188
205
200
211
187
230
198
194
210
190
202
229
LC-MS/MS
Protein number，Mr
Protein identification
MASCOT score
Accession number
Species
Biological function
P1,54.29
aldehyde dehydrogenase
418
gi|73760168
Sphingomonas sp. 14DN-61
PAH metabolism
P2,33.07
Biphenyl-2,3-diol 1,2-dioxygenase (23OHBP oxygenase)；
(2,3-dihydroxybiphenyl dioxygenase) (DHBD)
952
gi|115106
P3,47.167
large subunit of oxygenase
258
gi|28971830
Sphingomonas sp. P2
PAH metabolism
P4,33.07
2'-hydroxybenzalpyruvate-aldolase
299
gi|5578711
Sphingomonas xenophaga
PAH metabolism
P5,35.53
putative 2-hydroxy-benzylpyruvate aldolase
520
gi|28971850
Sphingomonas sp. P2
PAH metabolism
P6,36.93
4-hydroxy-2-oxovalerate aldolase
212
gi|73760166
Sphingomonas sp. 14DN-61
PAH metabolism
P7,32.99
putative acetaldehyde dehydrogenase
800
gi|28971838
Sphingomonas sp. P2
PAH metabolism
P8,22.42
putative 2-hydroxychromene-2-carboxylate isomerase
179
gi|28971822
Sphingomonas sp. P2
PAH metabolism
P9,21.03
putative glutathione S-transferase
419
gi|28971832
Sphingomonas sp. P2
PAH metabolism
P10,21.05
Chain A, Structure Of Sphingomonad, Glutathione S-Transferase
Complexed With Glutathione
446
gi|9257055
Sphingomonas sp. P2
PAH metabolism
P11,46.02
isocitrate dehydrogenase
70
gi|148553381
Sphingomonas wittichii RW1
PAH metabolism
P12,
20.79
Chain B, Crystal Structure Of The Terminal Component Of The PahHydroxylating Dioxygenase From Sphingomonas Sp Chy-1
611
gi|126030180
Sphingomonas Sp Chy-1
PAH metabolism
P13,53.17
ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit
156
gi|142082
P14,18.57
putative small subunit of toluene/benzoate dioxygenase
178
gi|28971828
Sphingomonas sp. P2
PAH metabolism
P15,18.57
small subunit of oxygenase
121
gi|28971824
Sphingomonas sp. P2
PAH metabolism
P16,33.28
1,2-dihydroxynaphthalene dioxygenase
181
gi|4335675
Sphingomonas xenophaga
PAH metabolism
PAH metabolism
Pyrene biodegradation by Strain
US6-1: Dioxygenation reaction
Pyrene biodegradation by
Strain US6-1:
Monooxygenation reaction
Applications in Bioremediation
Formulation of microbial consortium
a) Mixed substrates
Pyrene
BaP
PAHs remained (%)
100
 Strategy : Combined potent strains
with strain US6-1
 Strains : pyrene degraders, resin
degraders
 Condition : BaP, BaP + Pyr, OD=0.3
80
60
40
20
0
Control
US6-1 strain
US6-1+PAH
US6-1+Resin
PAH strains
Resin strains
Consortium
100
80
60
40
20
0
Control
US6-1 strain US6-1+PAH US6-1+Resin
Consortium
Results ;
 The biodegradation of PAHs mixture
was retarded by single use of strain
US6-1 but it was recovered by
combination of other strains
 Even in case of BaP only as a
contaminant biodegradation rate
was increased by combination of
US6-1 with resin degraders
b) Single (BaP) substrates
BaP remained (%)
Purpose ; Development of
effective consortium
Methods ;
PAH strains Resin strains
Purpose Lab test of strain US6-1 for
remediation of sediment contaminated
by PAHs
Methods
1013
7d
14 d
21 d
Total bacterial number (cells/g-dw)
1012
1011
1010
109
108
107
Sterilized Sterilized Control
Control
+ US6-1
control
+ US6-1
X Data
Control
12
Results
10
16 PAHs/d10-phenanthrene
 Sediment : collected from Gwangyang Bay
 Conditions :
 Controls : No treatment, Sterilized
 Inoculations : US6-1 on sediments,
on sterilized sediments
 Analysis : 16 PAHs by GC/MS
8
6
4
Control
Add US6-1
Sterilized control
Sterilized+US6-1
2
0
0
7
14
Time (Days)
21
 The inoculation did not affect the growth of
normal flora
 Sterilization enhanced the growth of
inoculated strain US6-1
 The inoculation accelerated the initial
process of degradation
Experimental procedure
- Contaminate 1 g oil on 50g
Sand or 100g Pebble
- Inoculate microbial agents with
inorganic nutrients
- Incubate at 4 ℃ or at room
temp.
- Measure the amount of evolved
CO2
Effect of 9 remediation agents on the mineralization of oil under
mild temperature
250
90
Sand (Mild Temp.)
Pebble (Mild Temp.)
80
200
70
CO2 (mM)
CO2 (mM)
60
150
100
50
40
30
20
50
10
0
0
0
2
4
6
8
Time (Days)
Time (Days)
U-blank (oil + sample)
6
4
2
0
8
A
B
C
D
E
F
G
H
S-blank (oil + sample + nutrients)
I
US6-1:included in A,H
G: no bacteria,functioned
as biostimulation
Effect of 9 remediation agents on the mineralization of oil under
low temperature
18
12
Sand (Low Temp.)
Pebble (Low Temp.)
16
10
14
8
CO2 (mM)
CO2 (mM)
12
10
8
6
4
6
4
2
2
0
0
0
1
2
3
4
5
6
7
4
2
0
8
Time (Days)
Time (Days)
U-blank1
S-blank1
A
B
C
D
U-balnk2
S-blank2
E
F
G
H
U-blank (oil + pebble) 3
S-blank (oil+peb+N.) 3
I
6
8
Conclusions
 Novosphingobium pentaromativorans US6-1 is capable of
degrading a wide variety of HMW-PAHs including pyrene,
benz[a]anthracene, chrysene, benz[b]fluoranthene and
benzo[a]pyrene.
 Degradation of pyrene in N. pentaromativorans US6-1 proceeds
via multiple metabolic routes initiated by mono-(C-1,2 and C-4,5)
and dioxygenation (C-4,5) reactions, further degradation via either
o-phthalate pathway or salicylate pathway, both pathways were
subsequently entered tricarboxylic acid (TCA) cycle and
mineralized to CO2.
 These studies provides evidence for the potential application of
this organism for improved PAH bioremediation and is necessary
in order to design efficient and predictable bioremediation
procedures.
Thank you
for your
attention!