PowerPoint Presentation Geomicrobiology at DUSEL

1
Geomicrobiology of the Subsurface
Tom Kieft Biology Department New Mexico Tech
2
Outline

• Subsurface geomicrobiology -- progress to date
• Witwatersrand Deep Microbiology Project
• Geomicrobiology at DUSEL
• Berkeley (Aug. 04) and Blacksburg (Nov. 04)
  DUSEL meeting progress

3
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

4
Community Structure
Enrichments genes, enzymes
Membrane lipids
16Sr DNA
Function
Subsurface Microbial Biogeochemical Cycling
Isolates Archives
Sampling
Environment
Microscopy Mineral Geochemistry
Dissolved Gases, Cosmogenic Stable Isotopes
Aqueous Geochemistry
5
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
6
EarthLab
7
Life in the slow lane!
8
(No Transcript)
9
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

10
Evander
3.0 Ga basement 2.0 Ga meteorite impact Uplift 2
km at 90 myr
Deep, sequestered microbial communities?
11
2.0 Ga
2.0 Ga
300 Ma
120oC
2.9 Ga
2.3 Ga
2.7 Ga
Basement 3.4 Ga
Geothermal gradients
25oC/km
9-15oC/km
20oC/km
12
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

13
Gas analysis of fissure waters

• CH4 (30-80)
• C2 (3-4)
• H2 (up to 30)
• He (up to 10)
• balance N2
• some NH3?

14
Geological Cross Section from West Driefontein
to East Driefontein

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

• 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

15
Service, dolomite, and fissure waters pe vs. pH
16
LMWL
Mean Precipitation
Service Water
Ancient Water
Deep Hydro- thermal Water?
Hot Springs
17
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
Novel Bacterial lineages unique to the SA
deep-subsurface South Africa Subsurface
Firmicutes Groups (SASFiG)
SASFiG-6
SASFiG-5

SASFiG-4
SASFiG-7
SASFiG-3
SASFiG-9

SASFiG-8
SASFiG-1
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-2
18
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

19
Why we need DUSEL for geomicrobiology

• Need for a dedicated site, with continuous
  long-term access, infrastructure, etc.
• Access to great depth (gt3 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.

20
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?

21
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?

22
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 (gt 1 Ma, preferably
  gt100 Ma)

23
More technical needs, desires, etc.

• Flowing water samples and/or core from deep sites
  (gt1 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
  (gt1 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.

24
Technical requirements for geomicrobiological
sampling

• tracers
• Solute Br-, fluorochromes (e.g., rhodmine),
  perfluorinated hydrocarbons
• Particulate fluorescent carboxylated 1-µm
  microbeads
• core diameters gt2 inches preferred
• drilling methods are highly site specific.
• anaerobic glove bag
• core barrels should be steam cleaned, core barrel
  liners
• freezer

25
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

26
The End
27
(No Transcript)
28
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 lt 20 kJ/mole
29
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?

30
Carbon leader gold deposit
31
Carbon leader contains uranium and organic carbon
along with gold
Carbon Leader
Phosphorimage
32
H2 generated by radiolysis of H2O and consumed by
microbes
Li-Hung Lin, Princeton
33
Fischer-Tropsch Synthesis of CH4 etc.
Typical thermogenic gas
Kloof Mine gas
Abiogenic gas from Canadian Shield
Sherwood-Lollar, Univ. Toronto
34
Sampling from a flowing borehole
35
6-pack in situ enrichment device 6 different
porous media Connected to flowing borehole for
several weeks
36
Culture-independent analysis 16S rRNA cloning
Microbial community
16S rDNA amplification (PCR)
Vector ligation
DNA Extraction
E. coli transformation
Culturing of isolated colonies
PCR on inserts
Plating and selection of recombinants
Phylogenetic analysis
RFLP screening
Sequencing
37
Microbial Diversity in Deep Gold Mine Samples,
Assessed by 16S rDNA Analyses
Tom Gihring1, Duane Moser1, Li-Hung Lin2, Bianca
Mislowac2, T.C. Onstott2, James Hall2, Ken
Takai3, David Balkwill4, David Stahl5, Brett
Baker6, Sean McCuddy7, and Tom Kieft7
1Pacific Northwest National Lab, 2Princeton
University, 3JAMSTEC, 4Florida State Univ.,
5Univ. Washington, 6Univ. Calif., Berkeley, 7New
Mexico Tech
Euryarchaeota
a-Proteobacteria
ß-Proteobacteria
d-Proteobacteria
g-Proteobacteria
Firmicutes
Firmicutes
Actinobacteria
Crenarchaeota
FCBs, Thermus, etc.
38
Desulfotomaculum-like organism (DLO)
unculltivated sulfate reducer spore-former
up to 95 of clone libraries genomic
sequencing in progress
39
S. African Au Mine Bacterial Isolates
PHBs
Thermus scotoductus SA-01
Vesicles
Bacillus thermoaureus
Auriminutor transvaalensis
40
Could life have originated in the deep subsurface?
From Karsten Pedersen
41
Could early life have survived Hadean bombardment
in the deep subsurface?
42
Drilling for subsurface life on Mars?
43
Undergraduate Workshops 2002 2003 REU 2003 and
2004
44
Processing subsurface samples in an anaerobic
glovebag
45
Culture-Independent Community Analysis

• Extract all DNA
• Amplify rRNA genes (rDNA) using polymerase chain
  reaction (PCR) and universal Archaeal or
  Bacterial primers
• Clone genes in Escherichia coli
• Screen clones by restriction fragment length
  polymorphism (RFLP) analysis
• Sequence rDNA of unique clones
• Compare sequences to databases
• Construct phylogenetic trees

46
Life in the Subsurface is Microbial

• Microorganisms
• Bacteria
• Archaea
• Protozoa
• Fungi
• viruses

47
microorganisms
macroorganisms
48
Geomicrobiology of the Subsurface
Tom Kieft Biology Department New Mexico Tech
49
Geomicrobiology of the Deep Subsurface

• Tom Kieft
• New Mexico Tech

50
Technical requirements for geomicrobiological
sampling

• tracers
• Solute Br-, fluorochromes (e.g., rhodmine),
  perfluorinated hydrocarbons
• Particulate fluorescent carboxylated 1-µm
  microbeads
• core diameters gt2 inches preferred
• drilling methods are highly site specific.
• anaerobic glove bag
• core barrels should be steam cleaned before use.
  core barrel liners
• freezer

51
(No Transcript)