Download Topic 10

MICROORGANISMS OF THE
DEEP SUBSURFACE
Christie Han, Raymond Hui, and Derek Lee
 Metabolism, Adaptations
3. Sampling/Analytical Techniques
 Cultivation vs. Molecular
4. Subsurface Studies
5. Challenges
6. Why Care about the Subsurface?
 Future directions
SEMINAR OUTLINE
1. What is the Deep Subsurface?
2. The Subsurface Environment
WHAT IS THE DEEP
SUBSURFACE?
WHAT IS DEFINED AS DEEP?
Varies according to different disciplines
Arbitrary numbers
Microbiological definition
Hydrologic framework
REGIONS OF THE SUBSURFACE
LIFE IN THE SUBSURFACE
Intraterrestrial life can be found in
various depths
Hydrogen, methane, carbon dioxide
gases formed deep inside earth
Huge biomass of intraterrestrial
microbes
ENVIRONMENTS FOR
INTRATERRESTRIAL LIFE
Water is common
Large solid surface area-to-water volume
ratio
Mostly in anaerobic conditions
Exception: radiolysis of water
Consolidated sediments, unconsolidated
material
Temperature and water activity is
limiting factor
WHAT IS GOING ON DOWN THERE?
(THE THEORIES)
Origin of Life
Thomas Gold, astrophysicist: life
originated beneath the surface
Adaptation of microorganisms to grow
and metabolize under the earth
Thermophilic lithotroph
Possibility of surface microbe interaction
with subsurface
Metabolism?
HYDROGEN GENERATION
1. Reaction between gases in magma
2. Decomposition of methane to graphite
and hydrogen at 600 oC temperatures
3. Reaction between CO 2, CH 4, H 2O at
elevated temperatures in vapours
4. Radiolysis of water
5. Cataclasis of silicates under stress
6. Hydrolysis by ferrous minerals in mafic
rocks
THE SUBSURFACE
ENVIRONMENT
SUBSURFACE ENVIRONMENTS
Macrohabitats
Ancient salt deposits
Caves
Critical Zone
Marine sediments
Microhabitats
Community Structure
Distribution
ENVIRONMENTAL CONDITIONS
Nutrients
Oxygen
pH
Porosity
Radiation
Salinity
Temperature
Tectonic activity
Water
CRITICAL ZONE
 Prokaryotes
 Bacteria
 Archaea
 Eukaryotes
 Fungi
 Algae
 Protozoa
 Viruses
 Constraints: microhabitat size and water
availability
SURFACE VS. SUBSURFACE
METABOLIC RATES
Surface MR = 10 -3 to 10 -1 g C/g cell C/hour
Subsurface MR = 10 -7 to 10 -5 g C/g cell C/hour
METABOLISM
Photosynthesis-independent
Indigenous or imported nutrients?
Sedimentary C
H 2 or methane (earth’s centre)
Oxidation of organic matter coupled to
electron acceptors at slower rates
Mean generation time = thousands of years!
TERMINAL ELECTRON
ACCEPTING PROCESSES (TEAP)
TEAPS (CONT’D)
PHOSPHOLIPID FATTY ACID
(PLFA) ANALYSIS
Quantitatively measures:
Abundance and distribution
Viable biomass
Community composition
Nutritional/physiological status
PLFA = viable; DGFA = non-viable
Are subsurface bacteria less resistant
to UV radiation than surface bacteria?
ADAPTATIONS
Surprisingly comparable UV
resistance as surface microbes
Critical conservation of DNA repair
pathways
Chemical insults e.g. oxygen radicals
Physiological characteristics
Pigmentation, cell wall thickness
ARE THEY ASLEEP?
(BACTERIAL DORMANCY)
 Does not arrest DNA degradation or protect
cellular components from chemical/radiolytic
insults
 Maintaining low MR and high DNA repair
capability is a superior strategy for the longterm
 Ribosomes and cell walls detected by FISH
ADAPTIVE METABOLIC
STRATEGIES
Sporadic growth
Slow growth rates
Periods of dormancy
 Adaptation to habitat variability
SAMPLING AND
ANALYTICAL
TECHNIQUES
EXTRACTION AND SAMPLING
Main method of extraction: Drilling
Samples must be properly extracted to
avoid contaminants
Major contaminant is drilling fluid
Sterility of core samples confirmed by
testing core samples for the presence of
drilling fluid
ANALYTICAL TECHNIQUES
Cultivation Dependent
 Direct count of
Organisms
 Growing of the
Microorganism
 Biochemical Activity
Cultivation Independent
(Molecular)
• RNA analysis
• Denaturing gel
electrophoreses
• RFLP
• FISH analysis
• More….
MOLECULAR TECHNIQUES
CG content analysis
DNA homology
RNA analysis
- probes
- 16S rRNA
- in situ Hybridization
Genomics, Metagenomics and Proteomics
Problems and Complications
STUDIES OF THE
SUBSURFACE
NEW DNA EXTRACTION METHOD
Under Construction…
SUBSURFACE ARCHAEA
Archaea dominate the subsurface
Lower permeability of cell membrane
Energized membrane, lower energy costs
Mediate methane production and
consumption
SUBSURFACE ARCHAEA (CONT’D)
 Ancient Archaeal Group
 Deep-Sea Hydrothermal
Vent Euryarchaeotal Group
6
 Marine Benthic Group B
 Marine Benthic Groups A&D
 Marine Group I Archaea
 Marine Hydrothermal Vent
Group
 Miscellaneous
Crenarchaeotic Group
 South African Goldmine
Euryarchaeotal Group
 Terrestrial Miscellaneous
Euryarchaeotal Group
PROBLEMS WITH
CHARACTERIZATION
 Isotope-labelled cells did not hybridize with
Archaeal organisms
 Methodological difficulty of the technique
 Uncharacterized phylogenetic diversity
 Primer mismatches
 Unequal distribution between the groups
 Inaccurate representation of the Archaeal
groups
PROBLEMS WITH
CHARACTERIZATION (CONT’D)
 Suggests unsampled subsurface diversity!
FUTURE IMPLICATIONS
New primer combinations/designs
Many uncharacterized Archaea
Better integration of phylogenetic and
biogeochemical observations
CHALLENGES OF
STUDYING THE
SUBSURFACE
CHALLENGES OF STUDYING THE
SUBSURFACE
High possibility of contamination
Study of subsurface microorganisms
survival rate to UV radiation and
hydrogen peroxide
Inaccuracies in quantification
Classical culturing techniques unable to
describe the total microbial community
In situ and in laboratory disparities
PREVENTION OF CONTAMINATION
Clean drilling equipment
Aseptic containment of samples
Tracers in drilling fluid
Sample surrounding environment
Immediate on-site analysis
FUTURE DIRECTIONS
BIOREMEDIATION
Exploit microbial metabolism
Radioactive wastes in the subsurface
Ex. Pseudomonas spp. in Antarctica
used to metabolize xenobiotic
compounds
NUCLEAR WASTE DISPOSAL
No method for proper storage/disposal
Use subsurface microorganisms:
Stabilize, retard, and assimilate
Compared to other waste repositories,
bacteria tend to be the most prominent,
making subsurface MOs a possible area
to look into nuclear waste disposal.
EXTREMOPHILES AND
ASTROBIOLOGY
Limited growth and survival conditions
Understanding habitability of deep
subsurface can be extrapolated to
habitability of other planets and
Astrobiology
WHY CARE ABOUT THE
SUBSURFACE?
 Extrapolate subsurface studies to astrobiology
 Application to bioremediation
- degradation of phenol and aromatics
 Uncovering a vast range of Archaea and
Bacteria in deep marine subsurfaces and
further understanding of marine microbial life
 Industrial Applications:
- Oil extraction
- Disposal of radioactive wastes
- Energy reservoirs in sub-ocean floor
sediments (methane)
SUMMARY
Definition of “deep subsurface”
Theories
Environment, Metabolism, and
Adaptations
Molecular techniques > Cultivation
Archaea dominate the subsurface
Contamination is a major issue
Subsurface MOs have a wide range of
uses!
QUESTIONS?