Class Schedule by rt3463df

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									Deep Subsurface
 Microbiology
                      Outline
• What is deep subsurface microbiology?
  – Savannah River drill site


• Sampling and analysis
  – core drilling
  – radio-labelling


• Observations of deep subsurface microbes
• Assignment #4
Deep Subsurface Microbiology
• Deep aquifers hundreds or thousands of
  metres below surface

• Previously concerned with impacts of
  microorganisms on drilling and mining

• Recently investigated for purposes of microbial
  sampling as prime objective
Deep Subsurface Microbiology
• 1966: US DOE studies at Savannah River

• Drilling performed to maintain suitable
  conditions for microbial sampling
Savannah River Drill Site
     Savannah River Drill Site
• Overlies the Atlantic Coastal Plain
• Sample drill hole: determines stratigraphy
• Unconsolidated sediments to 300 m deep,
  underlain by crystalline metamorphic rock or
  consolidated mudstone
• Some sandy aquifers interspersed between
  clay and silt formations
                 Sampling
• Samples collected in autoclaved, stainless
  steel core liners
• Sampling container below drilling fluid
• Tracer dyes to check for leakage into cores
• Sediments removed from core liners in N2-
  flushed glove bag (preserve anaerobic
  conditions) within 30 min of sampling
Core Sampler
    Subsurface Microbiology
• Environments vary considerably with
  composition of surrounding rock
• Deep-living microbes pervade both oceanic
  and continental crusts, especially in
  sedimentary formations
• Survival up to 100oC
     Subsurface Microbiology
• Porous conduits for groundwater: range
  from oxygen-rich to oxygen-poor
• Metabolism changes with oxygen zone
           Sample Analysis
• Incorporation rate of radiolabelled (14C-)
  acetate into lipids
• Incorporation rate of radiolabelled (32P-)
  thymidine into DNA
• Aerobic/anaerobic mineralization of
  radiolabelled (14C-) acetate, glucose to CO2

• MPN of aerobic heterotrophs
            Acetate to    Thymidine to
Depth (m)     Lipids     DNA (cpm/day) MPN (log/g dry
            (cpm/day)                    weight)
   0          3x107          ++++            7
   45       2.45x104           -             4
   58         3x103            -             6
  140        9.3x103          ++             2
  205        3.5x102          +++            2
  257        4.3x102          ++             4
  264       2.54x102          nd             4
            Aerobic Activity (cpm/day    Anaerobic Activity
                     x 103)                (cpm/day x 103)
Depth (m)    Glucose        Acetate     Glucose      Acetate
   45          291            678         nd            nd
   59           720          165          nd           nd
  146           92            18          nd           nd
  205           49            12          35            7
  257           203           52          17           12
  261           864         1065         132          1036
              Observations
• Culturable bacteria in clay sediments 3-5
  orders of magnitude fewer than in shallow
  aquifers or surface soils

• Sandy water-bearing layers had highest
  counts, and greatest activities, even compared
  to clay layers nearer the surface

• Depth not necessarily limiting to growth and
  activity
         Population Studies
• Enumeration of coliforms, sulfate-reducers and
  methanogens

• Anaerobic metabolism measured by
  disappearance of lactate, formate and acetate,
  and production of methane and H2S

• Anaerobes found in deep subsurface layers,
  but sediments were not primarily anaerobic
  – aerobes 100 - 100,000 X more abundant!
         Anaerobic Growth
• Anaerobes presumably growing in anaerobic
  microenvironments, or were aerotolerant

• Most anaerobes were found in water-saturated
  sandy zones, associated with anaerobic
  degradation of acetate and benzoate, and
  production of methane
       Population Dynamics
• Number of coliforms dropped rapidly away from
  surface layers
• No fecal streptococci

• Suggests that subsurface harbours a persistent
  coliform population, rather than acute sewage
  contamination
    Subsurface Denitrification
• Acetylene blockage method used to detect
  denitrification activity
  – NO2 N2O N2
  – can measure N2O, N2 production during
    denitrification by gas chromatography
  – acetylene (C2H2) blocks N2O N2
  – compare samples incubated +/- C2H2
    Subsurface Denitrification
• All tested samples, at all depths, showed
  denitrification activity

• Activity decreased with depth from surface
• Activity highest in water-bearing sandy
  sediments than in clay sediments

• Addition of NO2 enhanced denitrification in
  samples down to 289 m deep
                 SLiMES
• Subsurface lithoautotrophic microbial
  ecosystems
• Between mineral grains of igneous rocks
                        Autotrophs, derive energy from
                        inorganic chemicals

                        Other microbes derive energy from
                        organic compounds produced by
                        autotrophs
 Bacteria 1.7mi Below Surface!
• Incorporated into sedimentary rock during
  formation - “starvation” diet for millions of
  years?
   – long-term independence from photosynthesis

• Enter through infiltrating groundwater from
  surface? (most likely, in cases of igneous
  rocks due to high T during formation)

• SLiMEs may provide organic C

								
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