Sampling and characterizing subsurface microbial communities

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Lecture 6

• Sampling and characterizing subsurface microbial
  communities

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Non-sterile sample collection
Unsaturated systems Normally, mud is injected
through drill stem to cool bit and drive
borehole cuttings up to surface Non-sterile
core is collected in drill stem, brought to
surface, subcored, and frozen until
analyzed
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Sterile sample collection
Unsaturated systems HEPA-filtered air
is injected through drill stem to cool
bit and drive borehole cuttings up to
surface Core is collected in drill stem,
brought to surface, subcored, and frozen
HEPA filtered air
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Saturated Systems
Hollow auger core
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Sampling the deep subsurface for microorganisms
Non-sterile core sample
region sampled for microbiology
core section
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Bacterial cell densities obtained from cores with
different degrees of saturation
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2
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Acridine orange direct count
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Peptone, Tryptone, Yeast Extract, Glucose
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Microbiology of subsurface

• Most soil bacteria have not been cultured in lab
  because we have not met all their nutritional
  requirements or environmental conditions required
  for growth
• Molecular methods are allowing detection of many
  new types of soil and subsurface bacteria
• PCR of 16s rRNA gene fragments, clone library
  construction, sequencing, and comparing with 16S
  rRNA database.

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Estimates of microbial cell density based on
total DNA content of soil
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Estimating cell density from DNA content
DNA is extracted from soil with ethanol, and the
absorbance of the extract measured at 260nm with
a spectrometer. An absorbance reading of 1.0 is
equivalent to 50 mg DNA/ml solution Determine
what amount of soil (grams) was used to extract
the DNA
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Sample
Phylogenetic characterization

Unique 16s rRNA genes
Sequencing
ATAGGA

Diversity
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Pristine Site Coupon Library
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Contaminated Site Coupon Library
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Effects of disturbance on community diversity and
structure
Clone diversity from
biofilms
Pristine site
Contaminated site
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RFLPs
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RFLPs
Clone libraries created using intact
biofilm
PCR on hematite
chips suspended in groundwater
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Phylogenetic tree showing how strains are related
stopped
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Community Structure on Different Mineralsfrom
Same Subsurface Site
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Describe in outline format method of Terminal
Restriction Fragment Length Polymorphism
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Activities of soil and subsurface microorganisms

• Essentially all steps of the various
  biogeochemical cycles
• nitrogen fixation
• organic carbon oxidation to carbon dioxide
• denitrification
• iron reduction
• sulfate reduction
• methanogenesis

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Aerobic respiration rates
CH2O CO2
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Summary

• New molecular techniques can be helpful in
  identifying and relating new phylotypes
• Terrestrial environments, including the
  subsurface, contain very high-diversity microbial
  communities.
• Subsurface microbial communities can be altered
  by contaminants

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Summary

• Soil microbial communities are important vehicles
  for cycling C,N,P, and S
• Activities of subsurface microbial communities
  are low relative to those in surface environments
• Rates of reactions decrease with depth in
  subsurface