LBNL MC252 Oil Leak Research Team Terry C. Hazen, 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, Andre Cortis, Christian Rinke, Tanja Woyke, Theresa Pollard, and Olivia U. Mason [email protected]http://vimss.lbl.gov/horizonwiki/
Transcript
LBNL MC252 Oil Leak Research Team
Terry C. Hazen, 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, Andre Cortis, Christian Rinke, Tanja Woyke, Theresa Pollard, and Olivia U. Mason
• On April 20, the Deepwater Horizon (DH)exploded for unknown reasons (rising, rapidly expanding methane?)
• The floating rig burned for 2 days killing 11 people and then sank, shearing the 21-inch riser pipe some 600 ft above the BOP
• There were 3 breaks in the riser pipe• Originally placed at 5,000 barrels, recent
estimates place the oil intrusion to the Gulf of Mexico (GoM) at 60,000 barrels per day, 54.1 million cubic feet of natural gas was flared daily, 4.9 million barrels
• The “spill” has surpassed the Exxon Valdez spill of 0.26 million barrels in 1989 and the IXTOC-I blowout of 3.33 million barrels into the GoM in 1979, IXTOC-I was the second largest marine spill in the world.
• Well capped beginning July 12, 2010, no oil input to GoM after July 15, 2010, Macondo well declared dead September 19, 2010.
• Macondo Oil is light crude• specific gravity of 850 kg/m3
(API gravity 37.2°)• 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
Oklahoma Heavy Crude
Light Macondo Crude
Presenter
Presentation Notes
10 gpm (100/1) Nalco recommends 60/1
Where the oil went?The Federal Interagency Solutions Group, Oil Budget Calculator Science and
Engineering Team (November, 2010)
Predictive models of
geochemistry/ecology
Predictive models of community function
Predictive models of cellular function
Oil Spill Systems Biology
Geochemical processes
Microbial Population Correlates of Geochemistry
Measurement of biomolecular activities
during process
Simplified laboratory
communities with similar function
Cellular Network
Analysis of Function
Molecular analysis
LBNL Systems Biology Approach
1. Microbial Community Structure and Function (in situ analyses)a) Berkeley Phylochip: relative densities of 50,000 taxa of bacteria, archaea, and protists.b) GeoChip relative frequency of 50,000 functional genes.c) Phospholipid and Proteogenomics relative concentrations of functional lipids and
proteins.d) Metagenomics and Metatranscriptomics (JGI) total community and functional structure
via DNA/RNA sequencing.2. Isolation and lab simulations for physiological characterization and rate
determinations a) Synchrotron (Advanced Light Source) FT-IR real-time analysis of petroleum
degradation.b) High pressure simulations – 2500psi and higher, temperature regulated.c) Microrespirometer – rates of biodegradation under as many as 30 conditions
simultaneously, monitored real-time.d) Phenotypic Microarray – rapid biochemical and characterization and biodegradation
rates with growth curves 5000 time course assays at once.3. Geochemistry for linking structure and function using systems biology approach
a) Stable isotope analysis of petroleum and daughter products (Center for Isotope Geochemistry) linking biogeochemistry to petroleum bioavailability and community and population
b) Trace metal, hydrocarbon, and limiting nutrient characterization.
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, Missions 1-9• 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 of Alabama, Florida State University, Gulf Coast Research Lab – USM, Rutgers University, University of Oklahoma, Penn State University, University of Georgia, Scripps, University of Tulsa)
Two fixed wavelength UV fluorometers (Quantech/Thermo Scientific) were employed in tandem to determine fluorescence intensity ratios (FIRs). One fluorometer was equipped with a pair of wavelength filters allowing excitation at 280 nm and emission at 340 nm. The second fluorometer was equipped with the same 280nm excitation filter and a longer (445 nm) wavelength. Two individual aliquots of 3 ml were transferred to either methacrylate or UV grade quartz cuvettes, and emission at 340 nm and 445 nm was recorded on the respective fluorometers. FIRs were then calculated from fluorescence intensity at 340 nm divided by intensity at 445 nm.
Deep Plume Bathymetry
Camilli et al. August 19, 2010, Sciencexpress
Sampling sites around the MC252 well head from May 25 to June 7, 2010
Alkanes by depth and distance from well head May 9, 2010 until June 19, 2010
Distance (km)
Dept
h (m)
Total n-Alkanes Compoundsn-Alkane concentration (µg/L)
020406080100120140160180200220240260280300
BTEX by depth and distance from well headMay 9, 2010 until June 19, 2010
Distance (km)
Dept
h (m)
Total BTEX CompoundsBTEX concentration (µg/L)
0102030405060708090100110120130140150
PAH by depth and distance from well headMay 9, 2010 until June 19, 2010
Distance (km)
Dept
h (m
)Total PAH Compounds
PAH concentration (µg/L)
0100200300400500600700800900100011001200
Oil biodegradation
Petroleum Microbe
Daughter Products
OxygenCarbon dioxide
pH
Temperature
Fertilizer Water
Daughter Products
Daughter Products
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
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
average plume average non-plume
to
ta
l li
pid
/m
l
Signature Lipid analysis
Biomass
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
average plume average non-plume
Community Composition
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 γ-proteobacteria significantly
enriched in plume
γ-proteobacteria enriched in oil plume
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)
phospholipids16S rRNA
Clone Library
Microbial diversity comparison
Consistent with the results of the cloning and sequencing efforts for these three samples from Hazen, et al (2010), BM58 and OV011 had low species diversity relative to OV003.
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Num
ber o
f OTU
s
Number of sequences
Pyrotag sequences
BM58
OV011
OV003
Microbial community compositionSingle cell genomics
82% 70% 4%
Summary of GeoChip 4.0 probe and sequence information by functional gene category
• > 400 functional gene categories• Universal standards to allow data comparison across different experiments & times
Fig. DCA analysis of the normalized signal intensity data for oil-contaminated samples and non-oil samples showing that the oil spill significantly affected the microbial community structure.
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
Ref
lect
ance
3 micronMicrobial cells and oil degradation
Wavenumber (cm-1)
Ref
lect
ance
Wavenumber (cm-1)
Oxidation signatures
SR-FTIR analysis of Surface Water SampleAlkane (C-H) heat map Protein (N-H) heat map
3 micron
Wavenumber (cm-1)
Ref
lect
ance
A typical spectrum of oil from MC252
Normalized intensity0.0 1.0
N-H amide A of protein
C-H C-H
Oxidation products (C=O) heat map
Thin oil film
Oxidation products (O-H) heat map
Bacteria and oil drop
Bright-field, 100X
Hydrocarbon Biodegradation Pathways to Degrade the Compounds Found in Crude
Oil by Marine Microbes
OH
OH
COOH
CH3 -C-SCoAO
R-CH2 -CH2 -C-SCoAO
OR-CH CH-C-SCoA
R-CH-CH2 -C-SCoAOOH
R-C-CH2 -C-SCoAOO
CoASH
R-C-SCoAO
NAD +H++ NADH +
H2O
FAD
FADH2
Fatty acyl CoA
Acetyl CoA
TCA CO2
n-Alkane
Alcohol + H2O
Aldehyde
Fatty acid
COOH
CH3
OH
OH
OH
Catechol
COOHNH2
OH
OHHOOC
CH3
COOH
CH3
OH
COOH
OH
COOH
OHOCH3
COOH
OH
COOH
OHOHHO
Protocatechuate
Ring fission
Slide courtesy of Dr. Ron Atlas
MC-252 alkane half-life (days) from field and laboratory with currents of 2 - 5 days to move 10 km from source.
All samples BDL CTD-fluorescence (<1 ppm) and chemical analysis for all petroleum hydrocarbons (<2 ppb) N=170
Methane Biodegradation
Kessler and Valentine et al., A Persistent Oxygen Anomaly Reveals the Fate of Spilled Methane in the Deep Gulf of Mexico. ScienceExpress January 6, 2011
all compounds
branched alkanes
total glycol cmpds
propylene glycol
(ethylhexyl)sulfosuccinate
Corexit, 4°C k day-1 0.021 0.071 -0.002 -0.017 0.068T 1/2 days 32.2 9.7 na na 10.1
Corexit, 25°C k day-1 0.011 0.083 -0.015 -0.044 0.027T 1/2 days 62.3 8.4 na na 25.9
0
1
2
3
4
5
6
0 3 5 7 10 12
mg/
L
day
bis(2-ethylhexyl)sulfosuccinate4°C
25°C
0
10
20
30
40
50
60
70
80
90
0 3 5 7 10 12
mg/
L
day
Glycol compounds 4°C
25°C
0
5
10
15
20
25
0 3 5 7 10 12
mg/
L
day
Branched alkanes4°C
25°C
Corexit Degradation -- mixed community of microbes cultured from plume depth, non-plume
water
• Glycol compounds do not show any degradation in the laboratory• Bis (2-ethylhexyl) sulfosuccinate shows more degradation at 4°C• Branched alkanes degrade about the same at 4°C and 25°C
Flocs - remains of plume?
GoM Dead Zones
From DiMarco et al., 2010
Presenter
Presentation Notes
Fig. 1. Near-bottom (0.5–1.0 m above bottom) dissolved oxygen concentrations during May (top panel) and July (bottom panel) 2005. Hurricanes Cindy and Dennis impacted the northern Gulf of Mexico 3–9 July. Note: hypoxic conditions were not found between 91°W and 92°W during the July cruise, presumably because of strong mixing conditions during the storms that weakened the pycnocline. Dots are station locations.
Input of Oil in North America
(Oil in the Sea III - National Academy of Science, 2003)
Images that will be hard forget!
• 11 men, 6,104 birds, 605 sea turtles, 97 marine mammals killed Deepwater Horizon as of 10/14/10, GoM (4.9 mbbl)
• 2,500 birds killed Cosco Busan, San Francisco (0.0013 mbbl)• 250,000 birds, 22 orcas, 2800 otters killed Exxon Valdez, Alaska (0.26 mbbl)• 20,000+ birds killed Amoco Cadiz, France (1.6 mbbl)• 10,000+ birds Killed Ixtoc I, GoM (3.3 mbbl)
Houston Chronicle – Monday, October 4, 2010
Current Research Priorities
1. Determine oil droplet size, kinetics and rates of biodegradation with Macondo oil and water from deep plume. Manuscript submitted.
2. Respirometer studies of Macondo oil and Corexit at 5°C with deep plume water. Done. Paper about to be submitted.
3. Shoreline (Beach Sand) biodegradation and community structure. Done. Paper in preparation.4. Corexit 9500 biodegradation rates. Done. Paper in preparation.5. Metagenome and single cell sequencing on deep plume and surface samples. Done. Analysis
in progress.6. Surface plume biodegradation and community structure. Done. Analysis in progress.7. Repeat sampling of previous plume sites, especially those with DO dip, monitor microbial
community changes returning to ambient – oil bioindicators. Done. Analysis in progress.8. Detailed in situ hydrocarbon analyses of biodegradation and mixing in the deep and at surface.
Done. Analysis in progress.9. BioTraps baited with Macondo oil set at 1500m, 1100m, 200m, and surface to determine in situ
rates of biodegradation and microbe colonization in different environments. Done. Analysis in progress.
10. Determine kinetics and rates of biodegradation at high pressure and 5°C with Macondo oil and water from deep plume. In progress.
11. Sediment sampling for oil/Corexit and microbial communities (anaerobic conditions). In progress.
12. Natural oil seeps near MC252, microbial and oil characterization with ecogenomics, isotope geochemistry, and systems modeling. Planned.
13. Long-term Marsh studies. Planned.
Oil-amended Bug Traps• BioSep® beads are 3 mm
diameter spheres, made of 25% DuPont aramid polymer and 75% powdered activated carbon, with porosity of 75%.
• The large surface area can be baited with a chemical of choice; in this case baited with MC252 oil, using unbaited beads as the controls.
• The beads are housed in PVC traps, shown here, which have 2 mm slits to expose the beads to the soil/water environment
Deep-Sea Sediment Cores Collected September – October RS
