LBNL MC252 Oil Leak Research Team
Terry C. Hazen, Olivia U. Mason, Eric A. Dubinsky, Todd Z. DeSantis, Gary L. Andersen, Yvette M.
Piceno, Navjeet Singh, Janet K. Jansson, Alexander Probst, Sharon E. Borglin, Julian L. Fortney,
William T. Stringfellow, Markus Bill, Mark S. Conrad, Lauren M. Tom, Krystle L. Chavarria, Thana R.
Alusi, Regina Lamendella, Dominique C. Joyner, Chelsea Spier, Jacob Baelum, Manfred Auer, Marcin
L. Zemla, Romy Chakraborty, Eric L. Sonnenthal, Patrik D'haeseleer, Hoi-Ying N. Holman, Shariff
Osman, Zhenmei Lu, Joy D. Van Nostrand, Ye Deng, Jizhong Zhou, Kelly Wetmore, Jennifer Kuehl,
Rachael Mackelprang, Cindy Wu, Jen Lim, Fran Reid, Joern Larson, Jenni Hultman, Susannah G.
Tringe James Han,
Tringe,
Han Patrick Chain,
Chain Christian Rinke,
Rinke Tanja Woyke,
Woyke Theresa Pollard,
Pollard Edward M.
M Rubin.
Rubin
http://vimss.lbl.gov/horizonwiki/
tchazen@lbl.gov
http://www.nola.com/news/gulf-oil-spill/deepwater-disaster/index.ssf
Hydrocarbon
Composition
Light Macondo Crude
•
•
•
•
•
•
Macondo Oil is light crude
specific gravity of 850 kg/m3
(API gravity 37.2°, Exxon
V ld 29°)
Valdes
typical for the Gulf of Mexico
oil reservoirs at 6,000 m
carbon isotope signature is 27.42 ‰ ± 0.10 δ13CPDB
35% evaporates within 2 days
in a wind tunnel
45% in 2 weeks
North Slope Heavy Crude
Where the oil went?
The Federal Interagency Solutions Group, Oil Budget Calculator Science and
Engineering Team (November, 2010)
Systems Biology Approach
ALS
ALSsynchrotron
imaging
Hydrocarbon/Stable Isotope/Nutrient,
Physical/Chemical analyses
Lipids
PLFA
RNA
Single cell
isolation
Metatranscriptomics (JGI)
Isolation/phenotypic
characterization, cell counts
Cloning & sequencing
Single cell sequencing
((JGI))
DNA
PCR 16S rRNA genes
Metagenomics (JGI)
GeoChip (UO)
PhyloChip
454 pyrotag sequencing
(JGI)
Research Vessels
R/V Gyre
R/V Brooks
McCall
R/V Ocean Veritas
R/V Ferrel
Missions and Sampling
•
•
•
•
•
•
R/V Ocean Veritas – May 25 to June 11, Missions 1-3
R/V Brooks McCall – May 29 to June 27, Missions 5-9
R/V Ferrel – July 3 – August 29
29, Missions 1
1-9
9
R/V Gyre – September 11 – October 20, Missions 1-5
Filtered samples for ecogenomics (1-20 L) -80°C
Fixed samples for direct cell counts, hydrocarbons, isotopes,
metals, and nutrient analysis (10-125 ml) 4°C
• Live samples for isolation and lab simulations (1-20 L) 4°C
(1.2 km to 384 km in all directions from well head, collaborations with:
University
U
es yo
of Alabama,
aba a, Florida
o da S
State
aeU
University,
e s y, Gu
Gulf Coas
Coast Research
esea c
Lab – USM, Rutgers University, University of Oklahoma, Penn State
University, University of Georgia, Scripps, University of Tulsa)
http://vimss.lbl.gov/horizonwiki/index.php/Main
p
g
p p
_Page
g
Sample collection & processing
Station BM58
Deep Plume Bathymetry
Camilli et al. August 19,
2010, Sciencexpress
Sampling sites around the MC252 well
head from May 25 to June 7, 2010
Oil biodegradation
Oxygen
Oxygen
Carbon dioxide
pH
Daughter Products
Microbe
Daughter
aug te Products
oducts
Temperature
Daughter Products
Petroleum
Fertilizer (N, P, Fe)
Water
Characteristic depth profiles for distances from the
well head and one non-plume site
(diamonds = cell density)
Dispersed MC252 plume and non-plume
parameters at 1099-1219 m
Plume
Non-Plume
P
Berkeley Lab PhyloChip3 detects 50,000 different
bacteria and archaea in one test
Location and intensity of fluorescence determines occurrence and
relative abundance of 16S rRNA genes.
951 subfamilies were detected in 62 bacterial phyla.
Only 16 subfamilies in -proteobacteria significantly
enriched in plume
-proteobacteria enriched in oil plume
Plume samples are distinct from Pre-spill and Non-plume
Pre-Spill samples from Dr. Hollibaugh, UGA
ANOSIM Results
Deepwater sites using nonmetric multidimensional scaling ordination
of Bray-Curtis distances (stress = 3.98 and 4.55, respectively).
Significantly different by permutational analysis of variance (p = 0.005
for both)
16S rRNA
phospholipids
Microbial community composition
(Pyrotag)
93%
80%
3.0%
Single Cell Sequence
Hydrocarbon Biodegradation Pathways to Degrade the Compounds Found in Crude Oil by Marine Microbes
Oil by Marine Microbes
CH3
COOH
n-Alkane
OH
Alcohol + H2O
COOH
OH
OH
OH
OH
Aldehyde
COOH
NH2
Fatty acid
Catechol
CH3
TCA
Ringg fission
FAD
Fatty acyl CoA
COOH
COOH
Acetyl CoA
O
CH3 -C-SCoA
OH
OH
HOOC
Protocatechuate
CH3
O
R-CH
R
CH2 -CH
CH2 -C-SCoA
C SCoA
CO2
OH
COOH
COOH
COOH
HO
OH
OH
O
R-C-SCoA
FADH2
O
R-CH CH-C-SCoA
H2O
CoASH
O
O
R-C-CH2 -C-SCoA
OH
O
R-CH -CH2 -C-SCoA
OH
OH
OCH3
OH
H + + NADH + NAD +
Slide courtesy of Dr. Ron Atlas
Summary of GeoChip 4.0 probe and sequence
information by functional gene category
No. of gene
categories
11
No. sequences
retrieved
No. of probes
designed
No. CDS
covered
15754
3349
5547
Bacterial phage
40
3644
1100
2083
Carbon degradation
33
21529
9033
13667
Carbon fixation
5
5252
1762
3398
Methane metabolism
3
9718
507
1677
Nitrogen cycling
17
47988
7552
17550
Phosphorus utilization
3
3783
1378
2261
Stress response
45
75305
21574
41033
Sulfur cycling
6
8078
3254
4461
Metal resistance
44
25277
9478
17575
Contaminant degradation
184
44220
17919
30361
Energy process
4
1762
862
1131
Virulence
13
16762
3732
7444
Others ( gyrB, bchY)
2
7830
2492
4226
410
286,902
83,992
152,414
Functional process
Antibiotic resistance
Total
• > 400 functional gene categories
• Universal standards to allow data comparison across different experiments & times
Hydrocarbon degradation genes are highly enriched
Functional Gene correlations with
oil hydrocarbons
Dominant bacteria at 1099-1219 m, SEM and
acridine orange stain inset with distance from
wellhead
BM58 10km, 1179m
BM54 1.3km, 1194m
Floc SR-FTIR spectral analysis
SR-FTIR analysis of Surface Water Sample
Protein (N-H) heat map
Alkane (C-H) heat map
Thin oil film
Reflectance
(
)
µ
µ
100
80
60
40
C-H
C-H
20
3800
3600
3400
3200
3000
2800
2600
2400
2200
Wavenumber
2000
1800
1600
1400
1200
1000
8
(cm-1)
0.0
100
Normalized intensity
1.0
80
60
Oxidation signatures
N-H amide A
off protein
t i
40
20
3800
3600
3400
3200
3000
2800
2600
2400
Wavenumber
2200
2000
1800
1600
1400
1200
1000
Oxidation products (C=O)
heat map
80
(cm-1)
A typical spectrum of oil from MC252
Distance (micrometers)=137 µm
Refleectance
Reeflectance
3 micron
3 micron
Microbial cells and oil degradation
g
100
80
60
40
3800
3600
3400
3200
3000
2800
2600
2400
2200
2000
1800
Wavenumber (cm-1)
1600
1400
1200
1000
80
Oxidation products (O-H)
(O H) heat
map
Bacteria and oil drop
Bright-field, 100X
MC-252 alkane half-life (days) from field and laboratory
with currents of 2 - 5 days to move 10 km from source.
Alkane Ratios by site
C26/C15
C26/C16
C26/C17
3 50
3.50
3.00
2 50
2.50
2.00
3.40
1.50
2 84
2.84
1.00
1.80
1.61
0.50
0 85
0.85
1.02
0.56
0.43
0.37
OV01005
OV01104
1.00
0.66
1.48
00.91
91
0 89
0.89
0.00
MC252
BM053104 BM054104 BM057104 BM058104
1km
1km
5km
10km
Systems biology “the omics”
Metagenomics and Metatranscriptomics
Emulsion PCR of samples
Extract microbial b l
DNA and RNA
Sheer DNA/RNA
Emulsion PCR
Non‐oil samples Sample Sample Sample
1
2
3
Metagenomic sequences
Metatranscriptomics reveals in situ
reveals in situ activity Sequencing with Illumina sequencing platform
Genes involved in hydrocarbon degradation
1
0.01
Proximal station metatranscriptome
Distal plume
Uncontaminated
Proximal plume
0.001
Tolu
uene
Arom
matic
Ethylbenz
nzene
Par
articulate metha
ane monooxygen
enase
Ring hydroxyla
R
ating
Cyclohexa
anol dehydrogen
enase
n-alk
kane
Benz
zoate
Cyclohexan
none
Aldehy
hyde dehydrogen
nase
0.00001
Alco
ohol dehydrogen
nase
0.0001
FattyA
Acid
Gen
ne relative abundan
ance
0.1
Dominant genes in the metagenome and metatranscriptome
code for cycloalkane and n-alkane (C6 to C20) degradation.
Oceanospiralles model
Alkanes
CRISPR regions
to protect from
phage predation
Metabolome (metabolic pathways)
O Glycan
biosynthesis
O Mannosyl glycan
biosynthesis
N Glycan
biosynthesis
Glycan Biosynthesis
and Metabolism
High mannose type
N glycan biosynthesis
Glycosphingolipid
biosynthesis
globoseries
Glycosphingolipid
biosynthesis
lact and neolacto series
Butirosin and
neomycin biosynthesis
Lipopolysaccharide
biosynthesis
Glycosaminoglycan
biosynthesis
chondroitin sulfate
Other glycan
degradation
Puromycin
biosynthesis
Biosynthesis of
ansamycins
Glycosaminoglycan
biosynthesis
heparan sulfate
[B] Glycosyltransferases
Nucleotide
Metabolism
Streptomycin
biosynthesis
Thiamine
metabolism
Purine metabolism
Starch and sucrose
metabolism
• Oceanospirillales possess genes that code for
chemotaxis and motility as well as those for
degradation of C6-C20 alkanes and
cycloalkanes.
l lk
• Monooxygenases decreased probably due to
the abundance of easy to degrade alkanes
initially.
• Oceanospirillales exhibited a rapid
chemotactic response to the presence of oil,
oil
aliphatic hydrocarbons, but not methane,
which was very abundant.
Glycosaminoglycan
degradation
Galactose
metabolism
[B] Proteoglycans
Caffeine
metabolism
Ascorbate and
aldarate metabolism
Glycosylphosphatidylinositol
(GPI) anchor biosynthesis
Glycosaminoglycan
Glycosphingolipid
biosynthesis
biosynthesis
Arachidonic acid keratan sulfate
ganglioseries
metabolism
Metabolism of
Cofactors and Vitamins
Folate
biosynthesis
beta Alanine
metabolism
alpha Linolenic acid
metabolism
Polyketide sugar
unit biosynthesis
Pyrimidine metabolism
Pentose and glucuronate
interconversions
Riboflavin
metabolism
Vitamin B6
metabolism
Tyrosine metabolism
Biosynthesis of vancomycin
group antibiotics
Amino sugar and
nucleotide sugar
metabolism
Pentose phosphate
metabolism
Fructose and
mannose metabolism
Biosynthesis of 12 , 14
16 membered macrolides
and
Phenylalanine , tyrosine and
tryptophan biosynthesis
Glycerophospholipid
metabolism
Linoleic acid
metabolism
One carbon pool
by folate
Phenylalanine
metabolism
Isoquinoline alkaloid
biosynthesis
Photosynthesis
antenna proteins
Phenylpropanoid
biosynthesis
[B] Phytochemical
Compounds
Lipid
Metabolism
Ether lipid
metabolism
Fatty acid
biosynthesis
Taurine and
hypotaurine
metabolism
Sulfur
metabolism
Glycolysis /
Gluconeogenesis
Biosynthesis of
siderophore group
nonribosomal
peptides
[B] Photosynthesis
Proteins
Fatty acid
metabolism
Biosynthesis of type II
polyketide products
Retinol
metabolism
Acridone alkaloid
biosynthesis
Photosynthesis
Inositol phosphate
metabolism
3 Chloroacrylic acid
degradation
D Alanine
metabolism
Benzoxazinone
biosynthesis
Glycine, serine and
Glycine
threonine metabolism
Anthocyanin
biosynthesis
Limonene and
pinene degradation
Monoterpenoid
biosynthesis
Selenoamino acid
metabolism
Propanoate
metabolism
Sesquiterpenoid
biosynthesis
Lipoic acid
metabolism
Terpenoid backbone
biosynthesis
Caprolactam
degradation
Methane
metabolism
Primary bile acid
biosynthesis
Clavulanic acid
biosynthesis
Nitrogen
metabolism
Cystein and methionine
metabolism
Citrate cycle
(TCA cycle)
Atrazine
degradation
Porphyrin and
chlorophyll
metabolism
Valine, leucine and
isoleucine degradation
C5 Branched
dibasic acid
metabolism
Carbon
fixation
Steroid biosynthesis
Insect hormone
biosynthesis
Lysine
degradation
Reductive
carboxylate cycle
(CO2 fixation)
Tetrachloroethene degradation
Pantothenate and CoA
biosynthesis
Cyanoamino acid
metabolism
Fatty acid elongation
in mitochondria
Butanoate
metabolism
Valine, leucine and
isoleucine biosynthesis
Synthesis and
degradation of
Ketone bodies
Biosynthesis of type II
polyketide backbone
Zeatin
biosynthesis
Ubiquinone and
other terpenoid quinone
biosynthesis
Phosphonate and
phosphinate metabolism
Pyruvate
metabolism
Flavone and
flavonol
biosynthesis
Indole alkaloid
biosynthesis
Tryptophan
metabolism
Energy
Metabolism
Peptidoglycan
biosynthesis
Carotenoid
biosynthesis
Flavonoid
biosynthesis
Amino Acid
Metabolism
Carbohydrate
Metabolism
Sphingolipid
metabolism
Biosynthesis of
unsaturated fatty acids
Isoflavonoid
biosynthesis
Biosynthesis
y
of
Other Secondary Metabolites
Novobiocin
N
bi i
biosynthesis
Glycerolipid
metabolism
[B] Lipids
D Arginine and
D ornithine
metabolism
Alanine , aspartate and
glutamate metabolism
Glyoxylate and
dicarboxylate
metabolism
Steroid hormone
biosynthesis
Glucosinolate
biosynthesis
Tetracycline
biosynthesis
1,4 Dichlorobenzene
degradation
Naphthalene and
anthracene
degradation
Diterpenoid
biosynthesis
Biphenyl degradation
Metabolism of
Terpenoids and Polyketides
Secondary bile acid
Secondar
biosynthesis
Carbazole
degradation
1,2 Dichloroethane
degradation
Fluorobenzoate
degradation
Oxidative
O
idati e
phosphorylation
Brassinosteroid
biosynthesis
2,4 Dichlorobenzoate
degradation
Cytochrome
Xenobiotics Biodegradation[B]
P450 Substrates
and Metabolism
Bisphenol A
degradation
Glutathione
metabolism
Styrene
degradation
Fluorene
degradation
Benzoate degradation
via CoA ligation
Arginine and
proline metabolism
gamma Hexachlorocyclohexane
degradation
Toluene and
xylene degradation
Benzoate degradation
via hydroxylation
[B] Cytochrome P450
Ethylbenzene
degradation
1,1,1 Trichloro
2,2 bis(4 chlorophenyl)
ethane (DDT) degradation
Tropane, piperidine and
pyridine alkaloid biosynthesis
Penicillin and
cephalosporin
biosynthesis
D Glutamine and
D glutamate
metabolism
beta Lactam
resistance
Metabolism of
Other Amino Acid
Biotin
metabolism
Nicotinate and
nicotinamide
metabolism
Deep-Sea Plume 7/27-8/26/10
5-310 km
All samples BDL CTDfluorescence (<1 ppm) and
chemical analysis for all
petroleum hydrocarbons (<2
ppb)
b) N=170
N 170
Community analysis of deep-sea
GOM samples
•
•
•
•
82 samples analyzed with
PhyloChip so far from
5/27/10 – 8/24/10
Nearly all plume samples
different from background
Microbial community in
several DO dip samples
with no detected
hydrocarbons similar to
plume communities
Some DO dip samples
collected 6-weeks after
well was capped still
similar to plume samples
17 samples from Science
paper have outlined symbols
DO dips measured after well
was capped are circled
Cluster Analysis of PhyloChip OTU abundance
from BioTraps incubated 28 Days
Corexit Degradation -- mixed community of
microbes cultured from plume depth, non-plume
water
6
bis(2‐ethylhexyl)sulfosuccinate
90
4°C
5
Glycol compounds
25
4°C
80
Branched alkanes
25°C
25°C
4°C
20
70
25°C
4
60
50
mg/L
mg/L
mg//L
15
3
40
10
2
30
5
20
1
10
0
0
3
5
day
7
10
0
0
12
(
total glycol propylene ethylhexyl)sulf
cmpds
glycol
osuccinate
0
0
3
5
all compounds
day
7
10
branched alkanes
12
3
5
day
7
10
12
Corexit, 4°C
k
T 1/2
day
days
‐1
0.021
32.2
0.071
9.7
‐0.002
na
‐0.017
na
0.068
10.1
Corexit, 25°C
k
T 1/2
day
days
‐1
0.011
62.3
0.083
8.4
‐0.015
na
‐0.044
na
0.027
25.9
• Glycol compounds do not show any degradation in the laboratory
• Bis
Bi (2-ethylhexyl)
(2 h lh l) sulfosuccinate
lf
i
shows
h
more degradation
d
d i at 4°C
• Branched alkanes degrade about the same at 4°C and 25°C
Hydrocarbon mineralization
Control (no addition)
Oil
Oil + Corexit
Corexit
Control (no addition)
Killed control
Killed control with oil + Fe
Oil
Oil + Corexit
Oil + Fe
F
Oil + Corexit + Fe
Degradation of Oil and COREXIT
Oil degradation
Oil
Oil + Corexit
Oil + Fe
Oil + Corexit + Fe
COREXIT degradation
Sediment Analyses
120 multi-core sediment samples (64 R/V Gyre and
56 R/V Ocean Veritas). 29% showed potential
indication of contamination by oil, qualitatively,
only
y 7 ((6%)) exceeded aquatic
q
life benchmarks all
located within 0.33‐2.7 km of the wellhead
Operational Science Advisory Team – Unified
Area Command (OSAT, 12/17/10) Federal
On-scene Coordinator U.S. Coast Guard
“Other than (sediments near the
wellhead and near shore tar mats) the
OSAT found:
• No deposits of liquid‐phase MC252 oil
in sediments.
• No exceedances of EPA’s Human
H lth benchmark
Health
b h
k in
i water.
t
• No exceedances of EPA’s dispersant
benchmarks.
g , no exceedances off the
• Since 3 August,
aquatic life benchmark for PAHs in
water that were consistent with MC252
oil.
• Since 3 August,
August no exceedances of the
aquatic life benchmark for PAHs in
sediment beyond 3 km of the wellhead”
Slicing frozen sediment cores
Flocs - remains of plume?
Grains with alkane (C H)
Grains with clay (Al Al OH)
1.0
SR‐FTIR analysis of sediment sample SE‐20101001‐GY‐LBNL1‐BC‐124‐core 3
100
CH
Sulfate minerals (S O)
Normalize
ed intensity
Tran
nsmittance (%)
Grains with cement and clinker phase components
Carbonate minerals ( COO )
Al‐Al‐OH
COO
A typical sediment spectrum sho in alkane cla
showing alkane, clay, cement‐ and clinker phase‐
like absorption features
Al O
S O
Si O
20
4,000 3,000 2,000 1,500 1,000
Wavenumber (cm‐1)
Tricalcium aluminates (Al‐O)
0.0
Orthosilicate minerals (Si
(Si‐O)
O)
Scale bar = 50 µm
Elmer’s Beach
•Oil degrading bacteria were found in high abundance
•Community
C
i structure changed
h
d spatially
i ll and
d temporally
ll
•Members of Rhodobacteraceae family seem to dominate in the high TPH beach samples.
Day 0
Alcanivorax str.
str Abu-1
Day 2
Day 7
GoM Dead Zones
From DiMarco et al., 2010
Input of Oil in North America
(Oil in the Sea III - National Academy of Science, 2003)
Houston Chronicle – Monday, October 4, 2010
http://academy.asm.org/index.php/colloquia-reports/faq-series/291-faq-microbes-and-oil-spills
http://vimss.lbl.gov/horizonwiki
horizonwiki//
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When you stand by the Ocean…”