Surface to Lower Biosphere Limit: Long-term Geobiology Reference Transect

Why Biology Needs a DUSEL

Duane P. MoserDuane P. Moser

Desert Research InstituteDesert Research InstituteLas Vegas, NVLas Vegas, NV

Outline:

• Insights and frustrations from prior work

• General concepts to incorporate into design

• Specific ideas for long-term reference transect

Why Long-Term Reference Transect and why DUSEL?

•Almost always sporadic samples of opportunity

• Excavations always done for other purposes

• Very limited capacity for repeat sampling

Learning from persistent challenges from past

The Witwatersrand Deep Microbiology Project (1997-2003)

TC Onstott and many, many others

SAGMCG1

SAG

MC

G2

Cre

n G

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p 1

b

Cre

n G

rou

p 1

cC

ren G

rou

p 2

"Su

bsu

rface" Gro

up

2

Cren Group3

KorarchaeotaYNPFFA

OPA3/4

Thermoprotei

OPA2

Met

hanom

icro

bia

Eukaroyotes

Bacteria

No

rth

am G

rou

p 1WS

A2

Th

ermo

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alo

bac

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ap

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1 FCG3

Methanobacteria

pMC2

"Sed A

rchaea 1"

FCG1

SAGMEG-1

SAGMEG-2

Archaeoglo

bi

FCG2

Methanococcales

Thermococci

0.10

Marine Group 1

16S rRNA Tree by Thomas Gihring

Long-term Biosustainability in a High-energy, Low-diversity Crustal Biome

Science: Accepted pending revisions

L-H Lin, P-L Wang, D. Rumble, J. Lippmann-Pipke, E. Boice, L. Pratt, B. Sherwood Lollar, E. Brodie, T. Hazen, G. Andersen, T. DeSantis, D.P.

Moser, D. Kershaw, and T.C. Onstott

Brett Tipple, 3.3 kmbls in Mpneng

Hole EB5Evander Mine

Why Long-Term ?

Service water

Major source of introduced organisms.

Primarily Proteobacteria:Comamonadaceae, Hydrogenophaga, Leptothrix,

Alcaligenes, Nitrosomonas, Rhodobacter, etc.

Drilling fluid

Divergent from service water.

Mostly ProteobacteriaComamonadaceae, Hydrogenophaga, Thiobacillus, Thauera, Pseudomonas, Acenitobacter, Alishewanella, etc.

Borehole fluid, 1 hour:

Most similar to the drilling fluid community.

Introduced community overprints indigenous community.

Primarily Proteobacteria

Borehole fluid, 48 hours:

Still primarily Proteobacteria

Borehole fluids, 30 days:

Drilling fluid and service water communities no longer detected.

Desulfotomaculum and taxa deeply-branched Firmicutes appear.

Borehole fluids, 70 days

Population has stabilized.

7 taxa closely-related to Desulfotomaculum and deeply-branched Firmicutes.

drilling fluid

borehole fluid, 1 hour

borehole fluid, 48 hours

borehole fluid, 30 days

borehole fluid, 70 days

service water

unweighted arithmetic average clustering based on binary, presence/absence distance measures

Bacteroidetes

-Proteo.

-Proteo.

-Proteo.

Nitrospira

OP11

Firmicutes

Synergistes

Percent of clones

1006020

Bacterial 16S rDNA clone distribution

Microbial Community Development in Boreholes

South Africa Subsurface Firmicute Groups (SASFG)

*SASFG-1

SASFG-2

SASFG-3

SASFG-4

SASFG-5

SASFG-6

SASFG-7

SASFG-9

SASFG-8

image courtest of Gordon Southam

Major new bacterial lineages with one exception only found in South African subsurface below 1.5 km depth

Complete genome for SASFG-1 (LBNL). Sulfate reducing, spore former, motile, nitrogen fixer.

Tree by Thomas Gihring

Stable (Indigenous?) Populations

Dec-98

Feb-99

Nov- 2001

Nov-2002

Bacterial T-RFLP data “community 16S rDNA fingerprint (3.2 kmbls Driefontein)”

Isolate DR504

“SASFG-1”

Henderson Reference Transect

•Stable, predictable, platform

•Gold-standard reference site fortesting new technologies

•Deep ecological reserve

•Intact subsurface ecosystem

•“Artificial fracture”

•Track fluid movements (colonization history)

•Repeated sampling

In situ Experiments: Artificial Fracture Zone?

• Stevens and McKinley (H2 production in basalts) controversey… how important are fresh fracture surfaces and how fast do fault surfaces weather… do microbial communities respond to fault slip and other geological disturbances.

•Seismicity: do biofilms lubricate faults?

•Substrates (nutrient stimulation, recoverable mineral coupons)

Interface Between Oxic and Anoxic World

•Downhole packer

•Multilevel sampler

•U-tube with backfill

•Valve at outlet

Logistics (Hardware)

Operation at ambient pressure?

New systems from industry/DOE (e.g. oil, geothermal)?

• Steel Casings/Valves•Corrosion = failure (stainless?)•Iron source = shifts in population•Hydrogen artifacts

• Plastics/Rubber•PEEK, Delrin? (leaching?, degradation, pressure failure?)•Tubing (nylon, stainless)?

•Titanium?

Logistics (Materials)

• Distance•How far into the rock to escape mining influences?

• Drilling/Coring•Drilling muds (e.g. chemicals, bentonite, introduced bugs)•Rotary drilling with airlift?•Grout

• Legacy oxidation•Minerals oxidized during drilling•Steel cuttings remaining in hole

Logistics (Methods)

Biology DUSEL: Critical or Merely Important?

• Henderson DUSEL a unique opportunity to finally do subsurface microbiology “right”

• Long-term reference transect would be the gold-standard site for decades and adaptive to new technologies for life detection.

• Different hydrology/lithology at Henderson expands subsurface biomes that will have been explored

Conclusions

Description of experiment: a controlled platform for long-term geobiology laboratory, offering near-continuous coverage of an intact subsurface ecosystem block from shallow-aquifer to near the lower biosphere limit. the tracking of fluid migration in three dimensions and the testing of hypotheses concerning deep microbial colonization history. deep ecological reserve and gold-standard reference site, which could be sampled repeatedly over decades in response to new technologies.

Description of experiment: Roughly ten side-wall boreholes of a minimum 500 m length ea. would be extended horizontally at interval, and into hotter depths by drilling into the mine floor. Holes would be sealed to ambient pressure and outfitted with sampling ports, packers and unreactive multilevel samplers to allow repeated sampling proximal to features and host rock types of interest. Holes in unsaturated zones would be sealed and packered to enable gas sampling and down-hole collection of surface biofilms. Microbial population structure in the boreholes would be assessed using the best available molecular tools, both temporally from time-zero and spatially to quantify the extent and persistence of mining-induced contamination. Facilities would be developed to enable to emplacement and recovery of long-term in situ mineral weathering and substrate addition experiments.

Anaerobic Ecosystems: Life’s Redox Footprint(What would you expect in the very deep subsurface?)

Methanogenesis/Acetogenesis (consume H2)

H20 + CO2

Aerobic Respiration

Nitrate and Mn(IV) Respiration

Fermentations (release H2)

CH20 (Burial)

O2

Sulfate Respiration 1-1.5 nM

0

0.05 nM

H2 concentration

Fe(III) Respiration

7-10 nM

0.2 nM

1) No available respiratory electron acceptors?

A. Witwatersrand quartzite core from 1.95 km depth in fracture zone. Pink = rhodamine tracer. B. 35S auto-radiographic image of core. C. Sulfate reducing bacteria with AgS xtals in pore.

A.

B.

C.

Courtesy of Gordon Southam, Univ. of Western Ontario and Mark Davidson, Princeton University

Endolithic Sulfate Reducers(a shot in the arm for radiolysis)

Driefontein Consolidated Gold Mine

-Methananobacterium

-Actually an Archaeon (despite the name).

-Makes Methane from CO or CO2 and H2

-Desulfotomaculum

-Well known, sometimes thermophilic sulfate reducer

-Uses acetate, H2, probably CO

D8A microbial population

16S rRNA dsrA

mcrA

But wait a minute…..

Methanogens and sulfate reducers are not supposed to cohabitate!

30 M (radiolytic?) Sulfate

Vast excess (20,000 - 200,000 X) of abiogenic H2

An perfectly-poised, electron acceptor-controlled system?

Table 2. Free energy and steady state free energy flux for possible microbially-mediated reactions. Reactions were modeled frommeasured borehole water constituents.

Redox reaction Process

ΔG( /kJ mol)43 oC

p 9H .0

ΔG( /kJ mol)54 oC

p 9H .0

ΔG( /kJ mol)61 oC

p 7H .8

Power( /kJ c -ell s)

43 oCp 9H .0

Power( /kJ c -ell s)

54 oCp 9H .0

Power( /kJ c -ell s)

61 oCp 7H .8

1) 4C O + 5H2 O => CH4 + 3HCO3

- + 3H+Methanogen esis by

C O disproportionation-260 -257 -233 -8.6x10-14 -9.8x10-14 -1.0x10 -13

2) 4H2 + H+ + HCO3

- => CH4 + 3H2O

Hydrogenotrophi c methanogenesis -70 -63 -68 -6.6 10x -14 -6.9 1x 0-14 -8.6x10-14

3) 3H2 + C O => CH4 + H2O Hydrogenotrophi c methanogenesis -117 -113 -109 -3.9 10x -14 -4.3 10x -14 -4.9 1x 0-14

4) 4H2 + H+ + 2HCO3- =>

Acetat e + 4H2OAcetogenesis -48 -41 -46 -3.9 10x -14 -3.8 10x -14 -5.0 10x -14

5) 4C O + SO42- + 4H2 O =>

4HCO3- + HS- + 3H+ Sulf atereduction -297 -295 -271 -2.0 1x 0-14 -2.3 1x 0-14 -2.5 1x 0-14

6) C O + 2H2 O => HCO3- + H+ +

H2Wate -r shif t reaction -47 -48 -41 -1.6 10x -14 -1.8 10x -14 -1.8 10x -14

7) C O + H2 => 0.5Acetat +e 0.5H+ Acetogenesis -72 -69 -64 -2.4 10x -14 -4.2 10x -14 -1.2 10x -14

8) 4H2 + H+ + SO4

2- => HS- + 4H2O

Sulf atereduction -107 -102 -107 -7.2 10x -15 -7.9 10x -15 -9.7 10x -15

9) Acetat e + SO42- => 2HCO3

- + HS- Sulf atereduction -59 -61 -61 -4.0 10x -15 -4.7 10x -15 -5.5 10x -15

10) Propane+ 2.5SO42-+ 2H+

=> 3HCO3

- + 2.5H2S + H2OSulf atereduction -150 -153 -152 -4.0 10x -15 -4.8 10x -15 -5.5 10x -15

11) Aceta te + H2 O => CH4 + HCO3

- Acetoclastic methanogenesis -22 -23 -23 -3.9 10x -15 -4.6 10x -15 -5.4 10x -15

12 Ethan e + 1.75 SO42-+ 1.5H+

=> 2HCO3- + 1.75H2S +

H2OSulf atereduction -97 -100 -99 -3.7 10x -15 -4.4 10x -15 -5.1 10x -15

13) CH4 + S 4O 2- => H2 O + HCO3- + HS- Anaerobic methan e oxidation -37 -38 -38 -2.5 10x -15 -3.0 10x -15 -3.5 10x -15

CONTRIBUTORS

TC Onstott , Mark Davidson, Bianca Mislowack Princeton U

Jim Fredrickson, Tom Gihring, and Fred Brockman PNNL

Lisa Pratt, Eric Boice Indiana Univ.

Barbara Sherwood Lollar, Julie Ward, Greg Slater U of Toronto

Gordon Southam, Greg Wanger U of Western Ontario

Ken Takai JAMSTEC

Brett Baker UC Berkeley

Tom Kieft New Mexico Tech

Sue Pfiffner, Tommy Phelps U of Tennessee, ORNL

Dave Boone, Adam Bonin, Anna Louise Reysenbach Portland State U

Johanna Lippmann U of Potsdam

Terry Hazen , Eoin Brodie, et al. LBNL

Li-Hung Lin National Taiwan U

Dawie Nel, Walter Seymor, Colin Ralston, etc. etc. Mine professionals

Rob Wilson and staff Turgis Ltd. Consultants

Derek Litterhauer and Esta VanHeerden Univ. of Free State

Chrissie Rey, Faculty, students and staff U of Witwatersrand

2H/18O ratio and other chemistry matches other local waters aged to 3-30 MA

Hydrogen isotope equilibration temp = 60.5 oC e.g. 3 - 5 km source depth

Ca2+/Na+ ratio and other geochem indicates water has not traversed shallower levels (lavas and dolomites)

Thus water most likely aged meteoric, with long flow path, trapped in the Witwatersrand Supergoup (nearest outcrop = 11 km away.

The western Witwatersrand Basin

Ventersdorp lava (Ca2+/Na+ ratio 1.4 )

Witwatersrand quartzite (Ca2+/Na+ ratio 0.12 )

Dolomite (Ca2+/Na+ ratio 2.4 )1 km

2 km

3 km

4 km

5 km

6 km

54 oC temp is higher than geothermal gradient would predict (upwelling)

From Kelly, D.S. et al. 2005, Science, 1428-1434

Lost City (1, 3) Lidy Spring (2)

Columbia R. Basalt (3, 5) D8A (this work)

Marine Continental Continental Continental

Rock peridotite Basalt Basalt quartzite

pH 10 -11 6.9 7.5 - 9.5 9.1

CO2 low 55 mM 1 - 3 mM low

Temp 60 - 75 oC 59 oC 18 - 20 oC 54 - 61oC

CH4 1 - 2 mM 0.1 mM 2 - 209 M 17.5 mM

H2 <1 to 15 mM 0.0013 mM 0 - ca. 80 mM 0.165 mM

sulfate 1 - 4 mM 1.3 mM 0.004 - 1.4 mM 0.03 mM

Dominated Methanogen

SRB (firmicutes)

Methanogen Acetogen? Methanogen

Various SRBs

firmicutes

Methanogen

SRB (firmicutes)

1) Boetius, A. 2005. Science, 307:1420-14222) Chapelle, F. H., . et al. 2002. Nature 415:312-3153) Fry, N. K., J. K. Fredrickson, S. Fishbain, M. Wagner, and D. A. Stahl. 1997. Appl. Environ. Microbiol. 63:1498-1504.4) Kelly, D.S. et al. 2005, Science,307: 1428-14345) Stevens, T. O., and J. P. Mckinley. 1995. Science 270:450-454