Life in the Slow Lane:Life in the Slow Lane:Deep subsurface Deep subsurface geomicrobiologygeomicrobiology

Tom KieftTom KieftNew Mexico TechNew 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?

macroorganisms

microorganisms

Microbes couple oxidation of fuels

(electron donors) with reduction of oxidants (electron acceptors).

Subsurface Fuels:Microbes near the surface depend on photosynthetically generated organic carbon.

The deep biosphere may depend on geochemically derived energy sources: H2,

CH4, etc.

or < 20 kJ/mole

Subsurface microbiology 1986-present

• 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

SubsurfaceSubsurfaceMicrobialMicrobial

BiogeochemicalBiogeochemicalCyclingCycling

SamplingSampling

MicroscopyMicroscopy& Mineral & Mineral

GeochemistryGeochemistryAqueousAqueous

GeochemistryGeochemistry

EnvironmentEnvironmentDissolved Gases, Dissolved Gases,

Cosmogenic Cosmogenic & Stable Isotopes& Stable Isotopes

Membrane lipidsMembrane lipids

Isolates &Isolates &ArchivesArchives

FunctionFunction

16Sr DNA16Sr DNA

Community StructureCommunity Structure EnrichmentsEnrichmentsgenes, & genes, & enzymesenzymes

• 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 theslow lane!

Witwatersrand Deep Microbiology Project Team• 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.

South Africa

WitwatersrandBasin

Deep, sequestered microbial communities?

Evander

3.0 Ga basement2.0 Ga meteorite impactUplift ~2 km at 90 myr

2.0 Ga

2.9 Ga2.7 Ga

2.0 Ga 300 Ma

2.3 Ga

Basement 3.4 Ga

9-15oC/km25oC/km 20oC/km

120oC

Geothermal gradients:

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

• CH4 (30-80%)• C2+ (3-4%) • H2 (up to 30%)• He (up to 10%)• balance N2

• some NH3?

Geological Cross Geological Cross Section from Section from West Driefontein West Driefontein to East Driefonteinto East Driefontein

• Service water is then pumped 1-2 kilometers to the stopes• Where the carbon leader is mined

• To cool circulating air, control dust levels, and cool drilling equipment

• From the stopes, the now hot water flows to the base of the shaft and is pumped to the surface (red arrows)

• Where it is chilled, treated and recirculated to the subsurface

• Dolomite water drawn from the IPC pump chamber at shaft #4 augments supply

• Service water is chilled Service water is chilled to 4to 4°°C and treated with C and treated with chlorine and bromine chlorine and bromine before it descends shaft before it descends shaft #5 to mining levels #5 to mining levels (blue arrows)(blue arrows)

Service, dolomite, and fissure waters: pe vs. pH

Be339

Be341

Be23Be116GDW

Be24

DR938H3

Ev818-1,2,3

Ev219h5

E4IPC

E546bh1 W638bh1

WUD109

E5SW E5Sump

KL441SWKL441FW1HWDS

WDF1

WDF2&2b

B225FW1

Be116IDW

DR938 H1 110201

DR938H2 082001EV522FW030801EV821FW101601

EV914dFW

KL443FW050801

KL739062901

MBNWFW

Mean Precipitation

AncientWater

DeepHydro- thermalWater?

Service Water

LMW

L

Hot Springs

*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

* SASFiG-1

SASFiG-2

SASFiG-3SASFiG-4

SASFiG-5

SASFiG-6

SASFiG-7SASFiG-9

SASFiG-8

*image courtesy of Gordon Southam

Culture-dependent and culture-independent geomicrobiological characterization:Novel indigenous microbes and communitiesNovel and unusual deeply branched sequences may be indicative of ancestral linkages, (early life?), Novel products for biomed and biotech applications

Novel Bacterial lineages unique to the SA deep-subsurface:

South Africa Subsurface Firmicutes Groups (SASFiG)

1 m

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 long-term 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, U-rich 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– Sequence-specific

genetic probe

• Test for substrate utilization in the same cells by MAR or phoshorimaging– Uptake of 14C

substrate