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Robert Sanford
University of Illinois Urbana-Champaign
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Acknowledgements
Co-authors: Yiran Dong and Bruce Fouke (UIUC Dept. of Geology and Carl R. Woese Institute for Genomic Biology )
DOE, BP EBI and NASA funding.
Members of the Fouke lab.
Illinois State Geological Survey (ISGS), Midwest Geological Sequestration Consortium (MGSC), Schlumberger Limited
Roy J. Carver Biotechnology Center, UIUC
Dr. Joanne Chee-Sanford (USDA-ARS)
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Illinois Basin-Decatur Well Project:
an opportunity to explore the subsurface
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The IBDP is the first large-scale industrial carbon capture and storage facility in North America
Potential storage reservoir for more than 250 million tons of CO2 at depths of around 7,000 feet (2.1 km)
Excellent opportunity to study the geology, geochemistry, geophysics and geomicrobiology in a reservoir-like environment.
Two bore holes and wells installed in 2009 and 2011 provided opportunities to sample the Mt. Simon formation at different depths using the Quicksilver probe and Westbay systems, respectively.
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Key Questions:
What is the geochemical nature of the brines in the deep subsurface?
How might mineral surface features influence the microbial ecology?
How diverse are the microbial communities in the subsurface?
Can we cultivate microbial populations under their native environmental conditions?
What types of reactions are mediated by resident microbial populations?
Does the microbial community composition and physiologic potential vary spatially (in depth)? Is any variation coupled to known changes in geochemical and physical parameters?
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Illinois Basin
5http://sequestration.org/basin.htm
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Sampling History: Two wells
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5872 ft
2009
103-104
cell/L
5653 ft (1.8 km)2011
6632 ft (2.0 km)2011
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50 mm
Hematite/Goethite
Quartz
Sandstone
Formation Water
(NO3-, SO4
2-, TOC)
Dissolved Gas
(CO2 (aq), H2(aq))
Porosity
Calcite
overgrowth
Geologic/ Mineral Setting
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Geochemistry of Formation Watera
5655 6634
Eh
(mV/pH unit)b33.4 to -85 -33.2 to -82
pH (±0.3) 5.6 to 6.3 5.9 to 6.4
T (°C) 47 50
Density (g/mL) 1.102-1.100 1.135-1.137
Fe(II) (mM)c 1.19-1.51 1.28-1.37
TOC (mg C/L)d 56.5 55.4
TDS (g/L) 167 228
Total N (mg/g) 0.012 0.02
a Part of analyses were performed by ISGS. bT, Eh, pH and density were measured in-
situ at well head or in the trailer, while other parameters were measured on treated
samples in the lab; c Range for the results indicates difference between swab and
Westbay samples; d TDS was measured as the total solid content in freeze-dried
samples. 8
5655 ft (1.8 km) 6634 ft (2.0 km)
What kinds of microorganisms would we expect?
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Microbial Composition
0 20 40 60 80 100
5655
5872
6634
Percentage Composition (%)
Bacteroidetes
Firmicutes
Proteobacteria
SM2F11
Others
0 20 40 60 80 100
5655
5872
6634
Percentage Composition (%)
Orenia
Tepidibacillus
Halomonas
Other Others
ft (1.8 km)
ft (1.8 km)
ft (2.0 km)
ft (1.8 km)
ft (1.8 km)
ft (2.0 km)
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Halomonas sp.-5872 ft
pH: 5-10
Total dissolved solids: 0.5-
24%
Temperature: -1 to 35°C
Electron donors:
miscellaneous
Electron acceptors: oxygen,
nitrate
Genome: 4.2 Mb; 4166 genes
Halomonas sulfidaeris
Kaye et al., IJSEM, 2004; Gupta et al., unpublished
Closest known relative.
Conducted metagenomic sequencing of DNA from deep subsurface and used H. sulfidaeris genome as a scaffold to analyze the predicted metabolism of resident population.
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Actyl-CoA
Benzoate degradation
Benzoate
Benzoyl-CoA
Cyclohexane-1-
carboxylate
Crotonoyl-CoA
(S)-3-Hydroxy-
butanoyl-CoA
Acetoacetyl
-CoAAcetateFlagellum
Protein, aminoAcids
Fatty
acids
Sugars
Ribonu-
cleotide
Sidera-
phore
Ferric iron-
coated sandstone
Ox. Red
Iron
Reduction
ATP
ADP+Pi
ATP
synthesisF0F1-ATP
synthase
Formation water
Serpentin-
ization
Formate H2+CO2
HCO3-
HCO3-
Aromatic
compounds
Metals, toxin,
natural
antibiotics
Metals (e.g., Mn,
Zn, Ni,
Mo, Se,
Wo)
Fe(III)
Fe(II)Biosyn-
thesis
H+
Type IV
pili
H+ Na+/K+/
Ca2+
Nitrate
NO2-
Nitrate
Reduction
NH4+
Glutamine Glutamate
NH4+
Ammonia
Assimilation
Input signal
choline,
betaine
Ectoine
5-OH-ectoine
N-NAcDABA
DABA
N -NAcDABA
ASA
Asp
Choline
Betaine aldehyde
Glycine betaine
Osmosis
Response
Proteins/polysac
charide
K+AT
PADP
Restriction
modification
foreign
DNAs
MHost
DNA
M
Biofilm
ATP
PPhoB
?P PhoR
Urea
Purine Degradation
Allanoin
Allantoate
Ureidoglycolate
D-Glycerate
Pathway
Purine
Utilization
H+
Output signal?
GPD
G3PG3P
G6P
F6P
PGALD
PEP
G3P
pyruvate
TCA/rTC
A
6-P-gluconate
Phospholipids degradation
Aromatic compounds degradation
Succiny
l-CoA
Lipids
degradation
PPP ED
Acetyl
-CoA
Glycolysis
R
Metabolic
Reconstruction
Dong et al., Environmental Microbiology, 2014
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Enrichment and Isolation Efforts
0
10
20
30
40
50
60
70
0 5 10 15 20
Fe
(II)
/F
e(T
ot)
(%
)
Time (days)
0
10
20
30
40
50
60
70
0 5 10 15 20
Fe
(II)
/F
e(T
ot)
(%
)
Time (days)
AFH
Goethite
Hematite
Leptochrocite
Amorphous and crystalline ferric iron minerals were tested. (Pyruvate
(5 mM) Acetate (5 mM) H2 (5 mL/tube) Lactate (5 mM))
Hematite (Fe2O3) and goethite (a-FeO(OH)) have been identified as the
components of indigenous sandstone iron coating (Bowen et al, 2010)
5655 ft (1.8 km) 6632 ft (2.0 km)
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Isolation from Iron Reducer
from 5653 ft (where Orenia
dominated)
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Tepidibacillus decaturensis
strain Z9
Dong et al., Genome Announcement, 2016; Dong et al., IJSEM, 2016
pH: 5-8
Total dissolved solids: 1-5%
Temperature: 20-60 °C
Electron donors: sugars,
alcohols, VFAs and H2.
Electron acceptors: NO3-,
Fe(III)-citrate, ferrihydrite,
lepidocrocite, other oxidized
metals (e.g., MnO2)
Genome: 3.0 Mb; 2993 genes
0.5 mm
0 20 40 60 80 100
5655
5872
6634
Percentage Composition (%)
Orenia
Tepidibacillus
Halomonas
Other Others
NO3- NO2
- only
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Bacterial Isolate from 6632 ft.
Dominant organism from this depth
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Orenia metallireducens
(strain Z6)
Dong et al., AEM, 2016
pH: 5.5-9
Total dissolved solids:2-20%
Temperature: 20-60 °C
Electron donors: glucose (and
other sugars) and H2Electron acceptors: ferric iron
including amorphous ferric-
oxides and crystalline Fe(III)
minerals (hematite, goethite
and lepidocrocite)
Genome: 3.4 Mb; 3347 genes
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Comparison of Physiological Properties of
Iron Reducers with Closely Related Type
Strains
aL’Haridon et al, 2006; bOren et al. (1987 and 1991)
CharacterT. Decaturensis
Z9
Tepidibacillus
fermentansaO. Metallireducens
Z6 O. marismortuib
Habitat 1.7 km Illinois Basin,
IL
Underground gas
storage reservoir
2.1 km Illinois
Basin, ILthe Dead sea
Morphology Rod to filamentous
(0.3×3-10) Rod (0.3×(2-4))
Rod to Filamentous
(0.5×2-20)
Rod to Filamentous
(0.6×(3-13))
Gram Stain - + - -
G+C content (%) ~ 40 34.5 ~ 40 29.6
Oxidase - - - -
Catalase - - - -
NaCl range (g/L) 10-50 (25) 10-40 (10) 10-200 (30-120) 30-180(30-120)
Temp. range
(optimal) (°C)20-60 (30-40) 36-65 (50-52) 20-60 (30-50) 25-50 (36-45)
pH range
(optimal)5.2-8 (5.2-5.8) 5.5-8 (7.0-7.5) 6-9 (6-7) ND
Nutrient Source Fatty acids, sugars,
nitrate, heavy metals,
Fe-citrate, ferric iron
oxides
Fatty acids, sugars,
nitrate, no Fe
reduction
Sugars, nitrate,
heavy metals, Fe-
citrate, ferric iron
oxides, H2
Sugars, glycogen,
starch, no Fe-oxide
reduction
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Genomic and
Metabolomic
Reconstruction
of Fermentative
Iron Reduction
Pathway of
strain Z6
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New Concept for Fermentation
Facilitated by Microbial Iron Reduction
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Fermentation Reaction I: +
(22/15)H2O(4/3)CH3CH2OH + (2/3)CH3COO- + (2/3)HCOO- +
(4/5)HCO3- +(8/15)CO2+ (2/3)H2 + (32/15)H
+
Iron reduction-Reaction II: Fe2O3+4H++H2 +3H2O
[H+]fermentation-[H+]iron reduction≈0
Dong et al. 2017.. ES&T
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Summary Despite relatively similar physical and geochemical
characteristics, community composition varied with depth– shifting from Firmicutes dominated to Proteobacteria dominated.
This community heterogeneity with depth suggests that metabolic functions vary with depth.
Metabolism modeling based on metagenomic analysis yields helpful picture physiological potential.
Enrichment, isolation and genome sequencing efforts yield both dominant and non-dominant iron-reducing organism present in Mt. Simon formation and reveal a different metabolic reason to reduce iron; counter act acid generation.
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19
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Enhanced Fermentation by
Coupled Microbial Iron Reduction-
Orenia strain Z6
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21
(b)
(c) (d)
(a)
0
20
40
60
80
100
120
-45 -15 15 45 75 105 135
Re
ma
inin
g G
luc
os
e (
%)
Time (hours)
*
**
0
0.09
0.18
0.27
0.36
0.45
-45 -15 15 45 75 105 135
OD
600
Time (hours)
Control
Glucose+NaOH
Glucose
*
**
0
0.09
0.18
0.27
0.36
0.45
-45 -15 15 45 75 105 135
OD
600
Time (hours)
Control
Glucose+NaOH+Flushing
Glucose+Flushing
(e) (f)
**
*
*
*
*
*
**
*
**
4.5
5
5.5
6
6.5
7
-45 -15 15 45 75 105 135
pH
Time (hours)
4.5
5
5.5
6
6.5
7
-45 -15 15 45 75 105 135
pH
Time (hours)
0
20
40
60
80
100
120
-45 -15 15 45 75 105 135
Re
ma
inin
g G
luc
os
e (
%)
Time (hours)
pH adjustment pH adjustment+Gas Flush
-
22
0
20
40
60
80
100
120
-45 -15 15 45 75 105
Re
ma
inin
g G
luc
os
e (
%)
Time (hours)
*
*
0
1
2
3
4
5
6
0
0.09
0.18
0.27
0.36
0.45
-45 -15 15 45 75 105
Fe
(II) (mM
)
OD
600
Time (hours)
Abiotic control
Glucose+NaOH+Hematite
Glucose+Hematite
*
*
(a)
(b)
(c)
OD600 Biogenic Fe(II)
4.5
5
5.5
6
6.5
7
-45 -15 15 45 75 105
pH
Time (hours)
Fermentative Iron Reduction
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23
Genomic and
Metabolomic
Reconstruction
of Fermentative
Iron Reduction
Pathway of
strain Z6