Biodegradation of the Prestige
fuel oil under simulated
conditions
S. DÍEZa, J. SABATÉb, M. VIÑASb, J.
M. BAYONAa, A. M . SOLANASb and J.
ALBAIGÉSa
a
Instituto Investigaciones Químicas y
Ambientales de Barcelona-CSIC.
B Universidad de Barcelona. Departamento de
Microbiología
The fate of spilled oil in the marine
environment
Fuente: Albaigés 1989
OBJECTIVES
• To evaluate the biodegradation extent of
Prestige fuel oil in vitro experiments times by
two consortia.
– Aliphatics, aromatics and molecular markers.
• Effect of biodegradation on:
– source and weathering indices
• To assess weathering effects on
biodegradation.
• To evaluate the effect of oleophilic nutrients.
METHODOLOGY
Microbial Consortia Enrichment
Inocula: crude oil
contaminated
sand + 5-PAHs
(3-4 aromatic ring)
AM
5 years
Mineral medium
Inocula: diesel
contaminated
Soil + diesel oil
TD
Biodegradation Experiments
S200 : microemulsion urea + oleic acid + phosphate ester +2-butoxy-1-ethanol
original fuel
AM
50 mg
TD
50 mg
weathered fuel
AM
50 mg
TD
50 mg
fuel + S200
AM
50 mg
TD
50 mg
40 days
20 days
50 mg
original fuel
Chemical analysis
Chemical analysis
Chemical Analysis
Incubated fuel
surrogates
pH < 2.5
LLE (CH2Cl2)
organic extract
Fractionation
SPE (SiO2-C3CN)
Aliphatics
GC-FID
GC-MS
Aromatics
GC-MS
HYDROCARBON BIODEGRADATION
Biodegradation Aliphatics
22
15
100
original fuel
90
80
70
x
60
Ph
50
x
x
40
abundance
Pr
32
x
30
20
10
100
90
biodegraded fuel
20 days
80
70
60
50
Pr Ph
40
30
x
20
x
x
x
10
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
Time (min)
Biodegradation Aromatics
40
N1
35
original fuel
N2
30
N3
25
20
P P1 P2
15
P3
N
10
5
0
40
35
biodegraded fuel
20 days
30
25
20
15
10
5
0
2
4
6
8
10
12
14
16
18
20
22
24
Time (min)
26
28
30
32
34
36
38
40
42
44
3
2
1
2
C
3
BN
T
BN
T1
C
1
90
C
100
C
FL
FL
1
FL
2
FL
3
P3
P2
P1
P
D
D
D
D
3
BT
BT
3
N
N
C
ph 18
y
n- tan
al e
ka
ne
Al s
ky
lC
Al y
ky
lB
z
C
pr 17
is
ta
ne
Degradation (%)
Weathering Effect
AM40-original
AM40-weathering
80
70
60
50
40
30
20
10
0
3
3
2
1
2
1
3
BN
T
BN
T1
C
C
C
90
C
100
FL
1
FL
2
FL
3
FL
P3
P2
P1
P
D
D
D
D
BT
BT
3
N
N
C
ph 18
y
n- tan
al e
ka
n
Al es
ky
lC
Al y
ky
lB
z
C
pr 17
is
ta
ne
Degradation (%)
Strain Efficiency
TD40-weathering
AM40-weathering
80
70
60
50
40
30
20
10
0
3
2
1
3
BN
T
BN
T1
C
90
2
1
100
C
C
C
FL
1
FL
2
FL
3
FL
P3
P2
P1
P
D
D
D
D
3
BT
BT
3
N
N
C
ph 18
y
n- tan
al e
ka
ne
Al s
ky
lC
Al y
ky
lB
z
C
pr 17
is
ta
ne
Degradation (%)
Oleophilic Nutrient Effect
AM40-S200
AM40-original
80
70
60
50
40
30
20
10
0
Alkylbenzenes
100
(II)
ORIGINAL FUEL
m/z 106
alkyltoluenes
80
60
40
20
0
100
Relative Abundance
80
1-methyl-3-phytanylbenzene
60
(II)
BIODEGRADED (TD)
m/z 106
40
20
0
100
80
ORIGINAL FUEL
60
m/z 92
alkylbenzenes
40
(I)
20
0
100
80
phytanylbenzene
(I)
BIODEGRADED (TD)
60
m/z 92
40
20
0
6
8
10
12
14
16
18
20
22
24
Time (min)
26
28
30
32
34
36
38
C2 & C3-Phenanthrenes
100
C2-Phenanthrenes
(P2)
80
ORIGINAL
m/z 206
60
40
20
Relative Abundance
0
100
80
BIODEGRADED
60
40
m/z 206
20
0
100
C3-Phenanthrenes
(P3)
80
60
ORIGINAL
40
m/z 220
20
0
100
80
BIODEGRADED
60
40
m/z 220
20
0
16,0
16,5
17,0
17,5
18,0
18,5
19,0
19,5
Time (min)
20,0
20,5
21,0
21,5
22,0
22,5
23,0
C2 & C3- Dibenzothiophenes
100
C2- Dibenzothiophenes
(D2)
80
60
ORIGINAL
m/z 212
40
20
Relative Abundance
0
100
80
BIODEGRADED
60
m/z 212
40
20
0
100
80
ORIGINAL
C3- Dibenzothiophenes
(D3)
60
40
m/z 226
20
0
100
80
BIODEGRADED
60
m/z 226
40
20
0
13,5
14,0
14,5
15,0
15,5
16,0
16,5
17,0
17,5
Time (min)
18,0
18,5
19,0
19,5
20,0
20,5
21,0
21,5
Source Indices
4
100
Hopanes
m/z
90
1
2
3
4
5
6
191
3
80
70
60
5
2
50
27Ts
27Tm
29
30
32S
32R
6
40
30
1
20
10
0
27
28
29
30
31
32
Time (min)
33
34
100
m/z
10
217
80
9
7
Steranes
35
11
12
60
8
R
40
36
37
7
8
9
10
11
12
27dS
27dR
29S
29R
29S
29R
13
14
15
16
17
18
27R
27S
28R
28S
29R
29S
20
0
100
m/z
80
17
13 14
218
18
15 16
60
40
20
0
25
26
27
28
29
Time (min)
30
31
32
33
biodegradation
BIODEGRADATION MOLECULAR
MARKERS
Acyclic isoprenoids
Diasteranes
C27-steranes
-Steranes
Homohopanes
Monoaroamtic steranes
Triaromatic steranes
steranes hopanes isoprenoides
Effect of Biodegradation on
Source Diagnostic Ratios
Biodegraded oil
Index
Original oil
AM
20 days
AM+S200
40 days
Pr/Ph
0.98
0.97
---
%23
63
62
70
%24TT
11
11
7
%27Ts
22
21
22
%29
43
43
42
%27d
36
39
29
%29
S
45
49
49
%29
47
49
57
Source Indices
D2/P2
D3/P3
100
90
80
P2
70
60
P3
50
206+220
40
30
20
10
0
100
D2
90
80
D3
70
212+226
60
S
50
40
30
20
10
0
14,5
15,0
15,5
16,0
16,5
17,0
17,5
18,0
18,5
19,0
19,5
Time (min)
20,0
20,5
21,0
21,5
22,0
22,5
23,0
23,5
24,0
Effect of Biodegradation on
Source Diagnostic Ratios
Biodegraded oil
Index
Original oil
AM
20 days
AM+S200
40 days
D2/P2
34
34
25
D3/P3
40
37
33
Weathering Indices
D2/C2
D3/C3
C2
100
90
256+270
80
C3
70
60
50
40
30
20
10
0
100
D2
90
D3
80
212+226
70
S
60
50
40
30
20
10
0
13
14
15
16
17
18
19
20
21
22
23
Time (min)
24
25
26
27
28
29
30
31
32
Effect of Biodegradation on
Weathering Diagnostic Ratios
Biodegraded oil
Index
Original oil
AM
20 days
AM+S200
40 days
D2/C2
67
59
12
D3/C3
70
70
51
CONCLUSIONS
• N-alkanes, alkylbenzenes low MW parent PAHs and
mono and di-alkylated derivatives are degraded by
both consortia in 20-40 days incubation.
• Degradation rates of PAHs decrease according with
the alkyl substitution and number aromatic rings.
• Weathering slow down biodegradation.
• Oleophilic nutrient enhance the biodegradation of the
more refractory PAHs and some molecular markers.
• Source indicators can be modified when oleophilic
nutrient are added to the incubation medium.
ACKNOWLEDGEMENTS
• Financial support was obtained from the
MCYT (VEM2003-20068-CO5-01).
• Dr. S. Diez acknowledges a Ramon &
Cajal contract.
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Biodegradation of the Prestige fuel oil under simulated