The Subsurface Biosphere

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The Subsurface Biosphere

• Tom Kieft
• New Mexico Inst. of Mining and Technology
• T. C. Onstott
• Princeton University

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CHRONOLOGY OF SUBSURFACE MICROBIAL
INVESTIGATIONS
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Major accomplishments findings

• Tracer technologies
• Extended known limits of biosphere to gt 3 km
  depth
• Established biomass biodiversity
• Isolates in Culture Collections
• Correlated microbial activity with
• geological interfaces
• Rates of subsurface microbial activity
• Long-term survival of subsurface microbes

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NATURAL FLUID SUBSTRATE MIGRATION
Permeable Sedimentary Layers
HETEROTROPHY
DOC, H2 NH3
Nitrate
Pilansberg dyke
sulfate
Ventersdorp dyke
Vertical fluid transport along fault
DOC
ENERGY
DIC
DOC, H2 NH3
Contact metamorphic source
Fluid inclusions
N2
NH3?
CH4
CH4 filled fracture porosity
AUTOTROPHY
Deep crustal meteoric circulation
Deep crustal meteoric circulation
Mantle source
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LIFE IN THE EXTREMELY SLOW LANE
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(No Transcript)
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Build a team!
Community Structure
Takai-JAMSTEC
Balkwill-Florida State
Stahl-Univ. Wash. Baker-Univ. Wisconsin
PfiffnerWhite-Univ. Tenn.
FredricksonBrockman-PNNL
Function
Aseptic Sampling w/ Tracers
Subsurface Microbial Biogeochemical Cycling
Suflita-Univ. Oklahoma
Wilson-Turgis Tech. Alexander-Univ.
Wits. Spoelstra-Driefontein
DeFlaun-Envirogen Inc.
Boone-Portland State
Pedersen-Univ. Goteborg
Phelps-ORNL
Environment
Sherwood Lollar-Univ. Toronto
Pratt-Indiana Univ.
OmarHoek-Univ. Penn.
Southam-Northern Arizona
Stute-LDEO
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Community Structure
Function
Subsurface Microbial Biogeochemical Cycling
Sampling
Environment
Microscopy Mineral Geochemistry
Aqueous Geochemistry
Dissolved Gases Stable Isotopes
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2.0 Ga
2.0 Ga
300 Ma
Transvaal dolomitic sediments
120oC
10 kmbls
2.9 Ga
2.3 Ga
2.7 Ga
Basement 3.4 Ga
25oC/km
9-15oC/km
20oC/km
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ENERGY SOURCES AND CARBON SUBSTRATES
E5
CO2
H2
CH4
CH4
H2
CH4
MANTLE CH4 Hydrocarbons
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Witwatersrand Gases

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

Similar to Canadian and Fennoscandian Shield mines
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Sampling

• Rocks from freshly mined face
• Cores
• Fissure water from flowing boreholes
• biofilms

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Direct extraction, PCR and sequencing of 16SrDNA
from Au- and U-rich carbon leader yielded Thermus
and cyanobacteria? sequences
Carbon Leader
Phosphorimage
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How can you collect uncontaminated ore samples?

• SAMPLES CHISELED FROM FRESHLY BLASTED FACE
• LARGE BLOCKS COLLECTED FROM FRESHLY BLASTED
  RUBBLE
• SIDE WALL CORING INTO INACTIVE STOPE FACE

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• Microbiological, petrological, chemical and
• isotopic analyses of fracture fluid

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MINING IMPACT

• induced fluid migration
• biological contamination
• oxidation interface

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Service, Dolomite and Fissure water pe vs. pH
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gt1 Myr.
gt1 Myr.
Stute _at_ LDGO
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Dolomite
Acid Mine Water
Microbial sulfate reduction
Hi Sulfate Fissure Water
Sulfide Ore
Low Sulfate Fissure Water
Pratt _at_ IU
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Community Structure
Enrichments genes, enzymes
16SrDNA
PLFA-cell membrane
Function
Subsurface Microbial Biogeochemical Cycling
Isolates Archives
Sampling
Environment
Microscopy Mineral Geochemistry
Dissolved Gases, Cosmogenic Stable Isotopes
Aqueous Geochemistry
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West Driefontein Crenarchaeota
Kieft _at_ NMT
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Beatrix, W. Drie Kloof Fissure Water Sequences
W6
Acid Mine Service Water 16S rDNA Sequences
Takai _at_ JAMSTEC
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Environmental Clone OPB80 Group
Balkwill _at_ FSU
Anaerobaculum thermoterrenum Group
Fibrobacter and Acidobacterium
East Driefontein Bacteria
Low GC Gram-positive Bacteria
0.10
036
Desulfotomaculum halophilum
Desulfotomaculum ruminis
Desulfotomaculum luciae
Nitrospina Subdivision
Desulfotomaculum thermosapovorans
Flexistipes sinusarabici Assemblage
Fusobacteria and Relatives
unnamed organism AJ012599
Sporotomaculum hydroxybenzoicum
Gamma Proteobacteria
12/98 Clone Group 1 (18 clones
2/99 Clone Group 1 (27 clones
2/99 Clone Group 2 (2 clones)
Anaerovibrio burkinabensis
Sporomusa str. DR1/8
Beta Proteobacteria
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Desulfitobacterium frappieri
Thermoanaerobacter thermohydrosulfuricus
Thermoanaerobacter kivui
Alpha Proteobacteria
094
Bacillus thermocatenulatus
041
Flexibacter-Cytophaga-Bacteroides
Thermoanaerobacter kivui
Delta-Proteobacteria
High-GC Gram-positive Bacteria
Cyanobacteria and Chloroplasts
Planctomyces and Relatives
Environmental Clone OPB2 Group
Prosthecobacter Group
Spirochetes
Environmental Clone WCHB1-31 Group
Leptospirillum-Nitrospira Group
Nitrospira marina
Green Sulfur Bacteria
clone WCHB1-02
Green Non-Sulfur Bacteria Relatives
Thermus thermophilus
Deinococcus radiodurans
Thermotogales
Fervidobacterium islandicum
Petrotoga mobilis
Coprothermobacter proteolyticus Group
Coprothermobacter proteolyticus
Coprothermobacter platensis
Thermophilic Oxygen Reducers
Hydrogenobacter subterranea
Aquifex pyrophilus
Environmental Clone OPB45 Group
Thermodesulfobacterium hveragerdicum
clone OPB45
Archaea (outgroup)
Methanothermus fervidus
.10
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Complete 16S rDNA Sequence Analyses
Moser _at_ PUPNNL Takai _at_ JAMSTEC
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Pyrococcus in mine fissure water? - a
biological tracer of upwelling geothermal water?

• Fissure water characteristics
• Anoxic
• 60 C

• Pyrococcus abysii
• Found at deep sea hydrothermal vents
• Anaerobic
• Growth at 67-102 C
• Optimum 95 C

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Enrichments

• Fe(III), Mn(IV), NO3-, So, SO42- reducers,
• So Mn(II) oxidizers, methanogens, acetogens,
  methanotrophs, and fermenters.
• Samples inoculated aerobically/
• anaerobically, /-NaCl, /- H2 MPNs.
• Incubate up to 6 months at RT or 60oC.
• Transfer, isolate characterize growth
• vs. T, pH, salinity, radiation.
• 16Sr DNA archive.

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S. African Au Mine Bacterial Isolates
PHBs
Thermus multireducens
Vesicles
Bacillus thermoaureus
Auriminutor transvaalensis
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Thermus multireducens isolated from a West Rand
Au mine

• Reduces a variety of electron acceptors
• O2, nitrate, Fe(III), Mn(IV), S0, Co(III),
  Cr(VI), U(VI), Tc(VII)
• Thermophilic optimum temp. 650C
• Relatively radiation resistant
• D37 2000 Gy

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Auriminutor transvaalensis
Takai _at_ PNNL

• Extreme alkalophile optimum pH 11, range
  8-12
• Anaerobic, heterotrophic, fermentative metabolism
  with proteinaceous complex organic substrates as
  sole sources of carbon and energy
• yeast extract, peptone, and casein
• no growth on organic acids, alcohols etc.
• elemental sulfur, thiosulfate or fumarate added
  as accessory electron acceptors improved growth
• growth with thiosulfate induced black
  precipitates-presumably iron sulfide

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Auriminitutor transvaalensis
New genus in low GC gram positives
Takai _at_ PNNL
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Bacillus thermoaureus

• Thermophilic 55-70oC
• Microaerophilic growth with nitrate reduction to
  nitrite anaerobic growth with nitrate reduction
  to ammonia
• no growth w/other anaerobic electron acceptors
• Tolerant of a wide range of PAHs and straight
  chain hydrocarbons but not as growth substrates
• Produces thermostable lipases and esterases
• Spore former
• Forms PHBs under low nitrogen, low oxygen
  conditions

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Phylogeny-new species?
BACILLUS THERMOAUREUS
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Biofilms form from weeping fractures

• Biofilm samples had high lipid biomass 1300 to
  6600 pmol/g (3x107 - 2x108 cells/g).
• PLFA profiles indicate a diverse microbial
  community.
• Biofilm lipid profiles contain a higher relative
  proportion of free fatty acids and unidentified
  fatty acids as compared to fissure water or
  carbon leader samples.
• Sulfate reduction in enrichment cultures under
  saline and non-saline conditions and at 60oC.

Pfiffner _at_ UT
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Sulfate Reduction _at_ 3.2 kmbls., 37C
SRBs grown at 60oC
16S rDNA DSR Desulfofotomaculum geothermicum
d34S of sulfate sulfide indicate 90 conversion
FeS drip with 108 cells/g
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Growth Conditions in situ- E5-46-BH1 borehole
isolation experiment

• Terminally branched saturates and branched
  monoenoic fatty acids increased over time.
• These fatty acids are indicative for sulfate-
  reducing bacteria .
• Monoenoic fatty acids decreased over time in
  later three samples (containing gt1pmol PLFA/ml
  total biomass).

Pfiffner _at_ UT
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Sulfite reductase gene expressed
Columbia R. Basalt
E5 46 Bh1
West Driefontein
Archaea?
Stahl _at_ UW
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Results to date

• Four new bacterial species and many new 16S
  rDNA sequences.
• Hyperthermophilic(?) Archaea may
• reflect deep circulating groundwater (gt1 Ma).
• Radiolysis is source of H2 at West
• Rand

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Further questions

• Are the hyperthermophiles really
• hyperthermophiles?
• Does autotrophy support
• subsurface biosphere?
• What is the source of NH3?
• What are the in situ microbial
• growth rates?