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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
AM TD
Inocula: crude oil contaminated sand + 5-PAHs(3-4 aromatic ring)
Inocula: diesel contaminated Soil + diesel oil
Mineral medium
5 ye
ars
Biodegradation ExperimentsS200 : microemulsion urea + oleic acid + phosphate ester +2-butoxy-1-ethanol
AM50 mg
AM50 mg
TD 50 mg
original fuel weathered fuel fuel + S200
C h e m i c a l a n a l y s i s
20
da
ys
40
da
ys
AM 50 mg
TD50 mg
TD50 mg
original fuel
50 mg
C h e m i c a l a n a l y s i s
Chemical Analysis
Incubated fuel
pH < 2.5LLE (CH2Cl2)
organic extract
FractionationSPE (SiO2-C3CN)
AliphaticsGC-FIDGC-MS
AromaticsGC-MS
surrogates
HYDROCARBON BIODEGRADATION
Biodegradation Aliphatics
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
10
20
30
40
50
60
70
80
90
100
10
20
30
40
50
60
70
80
90
100 15
32
22
Pr Ph
x x
x
x
x
x x x
original fuel
biodegraded fuel20 days
Time (min)
abu
nd
ance
Pr Ph
Time (min)
0 5
10
15
20
25
30
35
40
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44 0
5
10
15
20
25
30
35
40
N
N1 N2
N3
P P1 P2
P3
Biodegradation Aromatics
original fuel
biodegraded fuel20 days
Weathering Effect
0
10
20
30
40
50
60
70
80
90
100
Deg
rad
atio
n (
%)
AM40-original
AM40-weathering
Strain Efficiency
0
10
20
30
40
50
60
70
80
90
100
Deg
rad
atio
n (
%)
TD40-weatheringAM40-weathering
Oleophilic Nutrient Effect
0
10
20
30
40
50
60
70
80
90
100
De
gra
da
tio
n (
%)
AM40-S200
AM40-original
6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38
Time (min)
0
20
40
60
80
1000
20
40
60
80
1000
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
20
40
60
80
100
(II)
(I)
m/z 106
m/z 92
(I)
(II)
m/z 106 alkyltoluenes
m/z 92alkylbenzenes
Alkylbenzenes
ORIGINAL FUEL
ORIGINAL FUEL
BIODEGRADED (TD)
BIODEGRADED (TD)
1-methyl-3-phytanylbenzene
phytanylbenzene
C2 & C3-Phenanthrenes
16,0 16,5 17,0 17,5 18,0 18,5 19,0 19,5 20,0 20,5 21,0 21,5 22,0 22,5 23,0
Time (min)
0
20
40
60
80
1000
20
40
60
80
1000
20
40
60
80
100
Re
lativ
e A
bun
danc
e
0
20
40
60
80
100
ORIGINAL
BIODEGRADED
ORIGINAL
BIODEGRADED
m/z 206
m/z 206
m/z 220
m/z 220
C2-Phenanthrenes(P2)
C3-Phenanthrenes(P3)
C2 & C3- Dibenzothiophenes
ORIGINAL
BIODEGRADED
ORIGINAL
BIODEGRADED
m/z 212
m/z 212
m/z 226
m/z 226
C2- Dibenzothiophenes (D2)
C3- Dibenzothiophenes(D3)
13,5 14,0 14,5 15,0 15,5 16,0 16,5 17,0 17,5 18,0 18,5 19,0 19,5 20,0 20,5 21,0 21,5
Time (min)
0
20
40
60
80
1000
20
40
60
80
1000
20
40
60
80
100
Re
lativ
e A
bun
danc
e
0
20
40
60
80
100
Source Indices
25 26 27 28 29 30 31 32 33 Time (min)
0
20
40
60
80
100 0
20
40
60
80
100
13 14
15 16
17 18
10
11 12
7
8
9
7 27dS 8 27dR9 29S 10 29R 11 29S 12 29R
13 27R 14 27S 15 28R 16 28S 17 29R 18 29S
m/z 217
m/z 218
1
2
3
4
5 6
27 28 29 30 31 32 33 34 35 36 37 Time (min)
0 10 20 30 40 50 60 70 80 90
100
1 27Ts 2 27Tm 3 29 4 30 5 32S 6 32R
m/z 191
Hopanes
SteranesR
BIODEGRADATION MOLECULAR MARKERS
Acyclic isoprenoidsDiasteranes C27-steranes -Steranes Homohopanes Monoaroamtic steranes Triaromatic steranes
biod
egra
datio
n
Effect of Biodegradation on Source Diagnostic Ratios
Index Original oil
Biodegraded oil
AM
20 days
AM+S200
40 daysPr/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
%29S 45 49 49
%29 47 49 57
%27 37 37 7
hopa
nes
ster
anes
isop
reno
ides
Source Indices
206+220
14,5 15,0 15,5 16,0 16,5 17,0 17,5 18,0 18,5 19,0 19,5 20,0 20,5 21,0 21,5 22,0 22,5 23,0 23,5 24,0Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
212+226S
D2D3
P2
P3
D2/P2 D3/P3
Effect of Biodegradation on Source Diagnostic Ratios
Index Original oil
Biodegraded oil
AM
20 days
AM+S200
40 daysD2/P2 34 34 25
D3/P3 40 37 33
Weathering Indices
212+226S
D2D3
D2/C2 D3/C3
13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
256+270
C2
C3
Effect of Biodegradation on Weathering Diagnostic Ratios
Index Original oil
Biodegraded oil
AM
20 days
AM+S200
40 daysD2/C2 67 59 12
D3/C3 70 70 51
• 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.
CONCLUSIONS
ACKNOWLEDGEMENTS
• Financial support was obtained from the MCYT (VEM2003-20068-CO5-01).
• Dr. S. Diez acknowledges a Ramon & Cajal contract.
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