Robert Sanford

University of Illinois Urbana-Champaign

1

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)

2

Illinois Basin-Decatur Well Project:

an opportunity to explore the subsurface

3

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.

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?

4

Illinois Basin

5http://sequestration.org/basin.htm

Sampling History: Two wells

6

5872 ft

2009

103-104

cell/L

5653 ft (1.8 km)2011

6632 ft (2.0 km)2011

50 mm

Hematite/Goethite

Quartz

Sandstone

Formation Water

(NO3-, SO4

2-, TOC)

Dissolved Gas

(CO2 (aq), H2(aq))

Porosity

Calcite

overgrowth

Geologic/ Mineral Setting

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?

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)

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.

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

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)

Isolation from Iron Reducer

from 5653 ft (where Orenia

dominated)

13

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

Bacterial Isolate from 6632 ft.

Dominant organism from this depth

14

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

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

16

Genomic and

Metabolomic

Reconstruction

of Fermentative

Iron Reduction

Pathway of

strain Z6

New Concept for Fermentation

Facilitated by Microbial Iron Reduction

17

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

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.

18

19

Enhanced Fermentation by

Coupled Microbial Iron Reduction-

Orenia strain Z6

20

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

23

Genomic and

Metabolomic

Reconstruction

of Fermentative

Iron Reduction

Pathway of

strain Z6