Paul Adrian Iturbe Espinoza
October 10,
Monthly Oil Spill Incidents - 2015
Volume of oil spills / month (bbl)
http://www.shell.com.ng
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The once-pristine Niger Delta has been polluted for decades
Musat, 2005
Study microbial community structure and functioning during
oil degradation in soils.
Isolation and characterization of dominant oil-degrading
strains from Nigerian soils under different culture conditions.
SOIL
MEDIA pH
Rich
Poor
Rich
Contaminated
Poor
Uncontaminated
Rich
Poor
5
7
Plating on
Nutrient
agar
28 days of incubation
Shaking at 120
rpm /30°C
No soil
5
7
5
7
5
7
5
7
5
7
47.5 mL of
medium
5 g of soil
2.5 mL of oil
Purify DNA from precultures (MoBio
Power Soil DNA Isolation kit)
Primers 8f and 1512r were used to
amplify almost complete 16S rDNA
Sequence PCR products by using
23 ABI 3730XLs (Macrogen)
Best hit after the
BLAST search
OD (600 nm)
4,0
R5-014Ly
R7UO24Cw(bc)
R7-O14Lc
NCDO712
P5CO16Lw
KF147
3,0
2,0
1,0
0,0
0
OD600=0.1
4
8
Sampling
12
Time (h)
16
20
24
Sampling
Add 2 mL of the
hydrocarbon
measure
OD600
vortex for 2 min
5 mL of cells
(OD600=1) in
buffer PUM
Rosenberg et al, 1980
Wait 15 min
Pseudomonas aeruginosa
Sphingobium fuliginis
Rhizobium sp
E. coli
R7CO16Bb
R7-O14Sy
R7-O26Lw
P7UO26So
P7-O24Lb
P7CO26B
P5-O26Sy
P5UO15Bbc
R7CO15Sy
R5-O14Ly
Marker
R7-O14Lw
R7-O14Lc
P5CO16G
P7-O16Woc
R7UO24Clwbc
P7UO26Lg
SSOSw
Marker
R5CO24Gc
P5CO16Lw
R7UO16Vwc
P7UO26Sw
NENY
SSOSy
SSOLw
SSOIc
Marker
Cont. soil
R7CO16Bb
Gram stain
Hydrophobicity
84.68%
Cont soil
P7CO26B
86,16%
Uncont. Soil P5UO15Bbc
47,94%
R7C016Bb P7C026B P5U015B(bc)
% Hydrophobicity
100
80
Octane
60
R7C016Bb
P7C026B P5U015B(bc)
40
20
0
R7C016Bb
P7C026 B P5U015B(bc)
Hexane
Octane
Hexane
No soil
R7-O14Sy
Cont. soil
R7CO15Sy
Cont. Soil
P5CO16G
Gram stain
Hydrophobicity
6h
24
53.5% 83.5%
55.1 % 89 %
57 %
81.3 %
1 mL KPi
3 mL MES +
2.5 mL
MgCl2 buffer
of oil
100 L of the oil (top)
layer were plated on
Nutritive agar
Shaking at 120 rpm/30°C
Over night
50 mL of
distilled water
Streak spin oil
SSOIc
SSOLw
Gram stain
Hydrophobicity
6.7 %
11 %
Rhodococcus sp.
Patulibacter sp.
Pseudomonas aeruginosa
Kocuria rhizophila
Micrococcus yunnanensis
Pannonibacter phragmitetus
94,9%
88.0 %
86,2%
93.5 %
91.6%
71.1%
Three strains change their hydrophobicity in stationary phase:
Dyella sp.
Pandoraea vervacti
Sphingobium fuliginis
6h
18.9 %
7.1%
55.1 %
24h
57,28 %
31.23 %
89.05 %
No soil
P7-O24Lb
Hydrophobicity
24,98%
Uncont. soil
R7UO16Vwc
88.02 %
Poor media/pH 7/Uncont. soil
P7UO26SW
Hydrophobicity
94,86%
Rich media/pH 5/No Soil
R5-O14Ly
91.55%
Pseudoxanthomonas sp.
9.2 %
Sphingopyxis soli
15.1 %
Dyella sp.
18.9 %
Labrys portucalensis
10.1 %
Pandoraea vervacti
7.1%
Swaminathania salitolerans 9.7%
Pseudomonas guguanensis
25%
Rhizobium sp.
6.7 %
Ralstonia sp.
27.8 %
Rhizobium sp.
11 %
Georgenia sp.
11 %
Niabella thaonhiensis
Pseudomonas aeruginosa
Sphingobium fuliginis
Achromobacter xylosoxidans
Novosphingobium capsulatum
44.0 %
47.9 %
57.0 %
42.6 %
49.2 %
Day 1
Ralstonia sp.
Rhizobium sp.
Georgenia sp.
Novosphingobium capsulatum
Day 28
Sphingobium fuliginis
Swaminathania salitolerans
Pseudomonas guguanensis
Kocuria rhizophila
Micrococcus yunnanensis
Achromobacter xylosoxidans
Pandoraea vervacti
Pannonibacter phragmitetus
After 28 days of incubation dominant strains are different as
a result of a adaptive response to the change of conditions
contaminated soils + oil
Labrys portucalensis
Dyella sp.
Pseudomonas aeruginosa
Sphingobium fuliginis
Niabella thaonhiensis
Sphingopyxis soli
Pseudoxanthomonas sp.
Patulibacter sp.
Rhodococcus sp.
uncontaminated soils + oil
After long incubation (28 day) dominant strains from contaminated
soils take over strains from uncontaminated soils. Both group at
the same time take over dominant strains from only oil.
1. 25 strains associated with oil degradation were
identify by partial 16S rDNA gen sequencing and
were characterized by DGGE fingerprinting, Gram
stain and cell surface hydrophobicity test.
1. Pseudomonas aeroginosa and Sphingobium fuliginis
are the dominant isolated, both strains have high
hydrophobicity and are potential hydrocarbon
degraders.
- Rob van Spanning
- Martin Braster
- Herwig Bachmann
- David Brown
- Juami van Gils
- Madalina Maria Vita