Surface to Lower Biosphere Limit: Long-term Geobiology Reference Transect
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Surface to Lower Biosphere Limit: Long-term Geobiology Reference Transect Why Biology Needs a DUSEL. Duane P. Moser Desert Research Institute Las Vegas, NV. Outline:. Insights and frustrations from prior work General concepts to incorporate into design

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

Why Biology Needs a DUSEL

Duane P. Moser

Desert Research Institute

Las Vegas, NV

Outline: Reference Transect

  • 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? Reference Transect

Learning from persistent challenges from past

  • Almost always sporadic samples of opportunity

  • Excavations always done for other purposes

  • Very limited capacity for repeat sampling

The Witwatersrand Deep Microbiology Project Reference Transect


TC Onstott and many, many others

Cren Group 2 Reference Transect

"Subsurface" Group 2

Cren Group3

Cren Group 1c

Cren Group 1b


Marine Group 1





















Northam Group 1




"Sed Archaea 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

Why Long-Term ? Crustal Biome

Hole EB5

Evander Mine

Microbial Community Development in Boreholes Crustal Biome

service water

drilling fluid

borehole fluid, 1 hour

borehole fluid, 48 hours

borehole fluid, 30 days

borehole fluid, 70 days

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

Bacterial 16S rDNA clone distribution












Percent of clones

Borehole fluids, 30 days:

Drilling fluid and service water communities no longer detected.

Desulfotomaculum and taxa deeply-branched Firmicutes appear.

Borehole fluid, 48 hours:

Still primarily Proteobacteria

Borehole fluid, 1 hour:

Most similar to the drilling fluid community.

Introduced community overprints indigenous community.

Primarily Proteobacteria

Drilling fluid

Divergent from service water.

Mostly Proteobacteria

Comamonadaceae, Hydrogenophaga, Thiobacillus, Thauera, Pseudomonas, Acenitobacter, Alishewanella, etc.

Borehole fluids, 70 days

Population has stabilized.

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

  • Service water

  • Major source of introduced organisms.

  • Primarily Proteobacteria:

  • Comamonadaceae, Hydrogenophaga, Leptothrix, Alcaligenes, Nitrosomonas, Rhodobacter, etc.

image courtest of Gordon Southam Crustal Biome

South Africa Subsurface Firmicute Groups (SASFG)











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

Dec-98 Crustal Biome


Nov- 2001


Stable (Indigenous?) Populations


Isolate DR504

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

Henderson Reference Transect Crustal Biome

  • Stable, predictable, platform

  • Gold-standard reference site for

  • testing new technologies

  • Deep ecological reserve

  • Intact subsurface ecosystem

  • “Artificial fracture”

  • Track fluid movements (colonization history)

  • Repeated sampling

In situ Experiments: Artificial Fracture Zone? Crustal Biome

  • 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)

  • Downhole packer

  • Multilevel sampler

  • U-tube with backfill

  • Valve at outlet

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?

  • 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

Conclusions Crustal Biome

  • 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

Description of experiment: Crustal Biome 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: Crustal Biome 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 Crustal Biome

(What would you expect in the very deep subsurface?)


H20 + CO2

H2 concentration

Aerobic Respiration



0.05 nM

Nitrate and Mn(IV) Respiration

0.2 nM

Fe(III) Respiration

1-1.5 nM

Sulfate Respiration

Fermentations (release H2)

7-10 nM

Methanogenesis/Acetogenesis (consume H2)

1) No available respiratory electron acceptors?

A. Crustal Biome

Endolithic Sulfate Reducers

(a shot in the arm for radiolysis)

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.



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

  • Methananobacterium Crustal Biome

    • 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 Crustal Biome


16S rRNA


But wait a minute….. Crustal Biome

Methanogens and sulfate reducers are not supposed to cohabitate!

30 mM (radiolytic?) Sulfate

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

An perfectly-poised, electron acceptor-controlled system?


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


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

The western Witwatersrand Basin Crustal Biome

Dolomite (Ca2+/Na+ ratio 2.4)

1 km

Ventersdorp lava (Ca2+/Na+ ratio 1.4)

2 km

3 km

4 km

5 km

Witwatersrand quartzite (Ca2+/Na+ ratio 0.12)

6 km

d2H/d18O 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

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

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.

1) Boetius, A. 2005. Science, 307:1420-1422 Crustal Biome

2) Chapelle, F. H., . et al. 2002. Nature 415:312-315

3) 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: 1428-1434

5) Stevens, T. O., and J. P. Mckinley. 1995. Science 270:450-454

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