Life in the Slow Lane:
Deep subsurface
geomicrobiology
Tom Kieft
New Mexico Tech
Outline
• Subsurface geomicrobiology -- background
• Witwatersrand Deep Microbiology Project
• Deep Underground Science and
Engineering Laboratory (DUSEL)
Geomicrobiology
• Low-level counting applications
Life in the Subsurface is Microbial
• Microorganisms
– Bacteria
– Archaea
– Protozoa
– Fungi
– viruses
Why study subsurface microbes?
• Reveal unknown
metabolic capabilities and
ecosystems
• Applications of novel
microbes
• Bioremediation of
contaminated aquifers
• Understanding waste
repositories
• Analogs for life on other
planets?
microorganisms
macroorganisms
Microbes couple
oxidation of fuels
(electron donors) with
reduction of oxidants
(electron acceptors).
Subsurface Fuels:
or < 20 kJ/mole
Microbes near the surface
depend on photosynthetically
generated organic carbon.
The deep biosphere may
depend on geochemically
derived energy sources: H2,
CH4, etc.
Subsurface microbiology 1986-present
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Drilling and tracer technologies
Extended known biosphere to >3 km
Revealed biomass & biodiversity
Isolates in culture collections
Linked microbial activity with
geological interfaces
• Slow rates of subsurface microbial
activity
• Indications of autotrophic ecosystems
Community Structure
Membrane lipids
Sampling
16Sr DNA
Subsurface
Microbial
Biogeochemical
Cycling
Environment
Aqueous
Geochemistry
Microscopy
& Mineral
Geochemistry
Enrichments
genes, &
enzymes
Function
Isolates &
Archives
Dissolved Gases,
Cosmogenic
& Stable Isotopes
• Subsurface biomass was considered insignificant but is now recognized
as a major fraction of planetary biomass (greater than surface biomass?)
• Subsurface microbial populations are: diverse, active, unusual, possess
novel traits, represent an exploitable resource
EarthLab
Life in the
slow lane!
Witwatersrand Deep Microbiology Project Team
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T.C. Onstott - Princeton
Duane Moser, Tom Gihring, Jim Fredrickson - PNNL
Barbara Sherwood Lollar - Univ. of Toronto
Lisa Pratt - Indiana Univ.
Tom Kieft – New Mexico Tech
Susan Pfiffner - Univ. Tenn.
Tommy Phelps - ORNL
David Boone - Portland State Univ.
David Balkwill - Florida State University
Gordon Southam - Univ. Western Ontario
Johanna Lippmann - Lamont-Doherty Earth
Observatory
Ken Takai - JAMSTEC
Esta van Heerden, Derek Litthaur -Univ. Free State
David Boone - Portland State Univ.
David Balkwill - Florida State University
Gordon Southam - Univ. Western Ontario
Johanna Lippmann - Geoforschungzentrum
Ken Takai - JAMSTEC
Esta van Heerden, Derek Litthaur -Univ. Free State
Many others, especially students.
Evander
3.0 Ga basement
2.0 Ga meteorite impact
Uplift ~2 km at 90 myr
Deep, sequestered microbial communities?
2.0 Ga
2.0 Ga
300 Ma
120oC
2.3 Ga
2.7 Ga
2.9 Ga
Basement 3.4 Ga
Geothermal gradients:
25oC/km
9-15oC/km
20oC/km
Geomicrobiological sampling
• Rocks from freshly mined
surfaces
• Fissure water from
flowing boreholes
– Filtered to concentrate cells
• including massive filtering
(~10,000 liters)
– In situ enrichment devices
• Cores -- especially useful
for sampling rock matrix,
fractures
• Biofilms
Gas analysis of fissure waters
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CH4 (30-80%)
C2+ (3-4%)
H2 (up to 30%)
He (up to 10%)
balance N2
some NH3?
Geological Cross
Section from
West Driefontein
to East Driefontein
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Service water is chilled
to 4°C and treated with
chlorine and bromine
before it descends shaft
#5 to mining levels
(blue arrows)
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Service water is then pumped 1-2 kilometers to the stopes
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From the stopes, the now hot water flows to the base of the shaft and is pumped to the surface
(red arrows)
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Where the carbon leader is mined
To cool circulating air, control dust levels, and cool drilling equipment
Where it is chilled, treated and recirculated to the subsurface
Dolomite water drawn from the IPC pump chamber at shaft #4 augments supply
Service, dolomite, and fissure
waters: pe vs. pH
Mean Precipitation
E5Sump
E5SW
Ancient
Water
E546bh1
E4IPC
DR938H3
WUD109
KL441FW1HWDS
W638bh1
KL441SW
WDF1
KL739062901
EV522FW030801
EV821FW101601
DR938 H1 110201
DR938H2 082001
MBNWFW
EV914dFW
KL443FW050801
Ev219h5
WDF2&2b
Be116IDW
Ev818-1,2,3
Be341
Be339
B225FW1
Be116GDW
Be23
Be24
Hot Springs
Deep
Hydrothermal
Water?
Culture-dependent and culture-independent
geomicrobiological characterization:
Novel indigenous microbes and communities
Novel and unusual deeply branched sequences may be
indicative of ancestral linkages, (early life?),
Novel products for biomed and biotech applications
image courtesy of Gordon Southam
Novel Bacterial lineages
unique to the SA deepsubsurface:
South Africa Subsurface
Firmicutes Groups
(SASFiG)
SASFiG-6
SASFiG-5
SASFiG-4
SASFiG-3
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*SASFiG-9 (isolated)
Detected within a water-bearing
dyke/fracture at 3.2 Km depth.
strictly anaerobic; iron-reducer
optimal growth temperature = 60 oC
virgin rock temp = ~ 45 oC
1 mm
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SASFiG-7
SASFiG-9
SASFiG-8
SASFiG-1
SASFiG-2
Geomicrobiology at DUSEL
• Probe lower limit of
the biosphere.
• Test “geogas”
hypothesis:
ecosystems dependent
on geochemically
generated H2.
• Study adaptations for
long-term persistence
of microbial
communities
• Geologic interfaces
Why we need DUSEL for
geomicrobiology:
• Need for a dedicated site, with continuous longterm access, infrastructure, etc.
• Access to great depth (>3 km)
– Test limits of life, depth of biosphere
– Ecosystems based on H2, “geogas”
• Monitor human impacts on the subsurface
• Biotechnical applications
– In situ mining
– Bioremediation
– Novel enzymes, pharmaceuticals, etc.
What are the big research
questions?
• What energy and carbon sources are available in
the deep subsurface? Importance of “Geogas”?
What are the sources of H2? Rates of H2
generation? Independence from photosynthesis?
• Are these ecosystems suitable analogs for possible
subsurface life on other planets?
• Are there subsurface microbes and communities
that are selected for and adapted to the extreme
conditions of the subsurface?
• How has the metabolism of indigenous
communities influenced subsurface geochemistry?
More research questions:
• What are the in situ rates of metabolism?
• What adaptations do microbes have that enable
persistence for geologic time periods under
extreme conditions?
– Low nutrient flux, high temperature, extreme pH, high
pressure, etc.
• How do subsurface microbes maintain/repair
macromolecular structures?
• Do subsurface microbes represent early life on
earth?
Technical requirements, desires, etc.
• Access to multiple locations with varied geology
(could be single or multiple sites)
– Igneous, metamorphic, and sedimentary rocks
• Access to locations with geological interfaces,
geochemical gradients
• Access to pristine “green fields” (unmined,
unimpacted by mining)
• Access at multiple depths
• Access to a deep site (2-3 km) from which to
drill/core through 121 C isotherm.
• Access to ancient groundwater (> 1 Ma, preferably
>100 Ma)
More technical needs, desires, etc.
• Flowing water samples and/or core from deep sites (>1
kmbls) with mineralogy that may be conducive to
abiotic, geochemical generation of H2 (e.g., basalt,
serpentinized ultramafic rock, Fe(II)-rich minerals, Urich minerals).
• Flowing water samples and/or core from deep sites (>1
kmbls.) with evidence of biological sulfate reduction
(significant H2S in ground water) or methanogenesis
(significant CH4 in groundwater or measurable partial
pressure of CH4 in localized areas of mine atmosphere).
• Biofilms from tunnel walls.
Technical requirements for
geomicrobiological sampling:
• tracers
– Solute: Br-, fluorochromes (e.g.,
rhodmine), perfluorinated
hydrocarbons
– Particulate: fluorescent
carboxylated 1-µm microbeads
• core diameters >2 inches
preferred
• drilling methods are highly site
specific.
• anaerobic glove bag
• core barrels should be steam
cleaned, core barrel liners
• freezer
DUSEL Geomicrobiology:
Opportunities for new
technologies
• Down-hole instruments
• Improved sampling and analyses of the
geomicrobiology of rock-water interfaces
• Increased sensitivities for metabolic assays
• More sensitive geophysical approaches
• Metagenomics, proteomics, metabolomics
Applications of low-level
counting in geomicrobiology:
• Dating groundwater, minerals, etc.
• Radiotracer experiments
– Detecting and quantifying low rates of microbial
activities
– Imaging microbial processes
– (However: limitation is likely the purity of
commercially available radiochemicals, not counting
sensitivities).
Radiorespirometry
• Low-level counting might enable lower-level abiotic
controls, and thus greater sensitivity, but:
– need higher purity radiochemicals
– Non-biological oxidation of 14C organic substrates can occur -Mars Viking exp’t
Fluorescent in situ hybridization (FISH)
and microautoradiography (MAR) or
phosphorimaging
• Identify
individual
microbial cells
by FISH
– Sequencespecific genetic
probe
• Test for substrate
utilization in the
same cells by
MAR or
phoshorimaging
– Uptake of 14C
substrate