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Christie Han, Raymond Hui, and Derek Lee
MICROORGANISMS OF THE DEEP SUBSURFACE
1. What is the Deep Subsurface?2. The Subsurface Environment
Metabolism, Adaptations
3. Sampling/Analytical Techniques
Cultivation vs. Molecular
4. Subsurface Studies5. Challenges 6. Why Care about the Subsurface?
Future directions
SEMINAR OUTLINE
WHAT IS THE DEEP SUBSURFACE?
Varies according to different disciplinesArbitrary numbers
Microbiological definitionHydrologic framework
WHAT IS DEFINED AS DEEP?
REGIONS OF THE SUBSURFACE
Intraterrestrial life can be found in various depths
Hydrogen, methane, carbon dioxide gases formed deep inside earth
Huge biomass of intraterrestrial microbes
LIFE IN THE SUBSURFACE
Water is commonLarge 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
ENVIRONMENTS FOR INTRATERRESTRIAL LIFE
Origin of LifeThomas Gold, astrophysicist: life originated beneath the surface
Adaptation of microorganisms to grow and metabolize under the earthThermophilic lithotroph
Possibility of surface microbe interaction with subsurface
Metabolism?
WHAT IS GOING ON DOWN THERE?(THE THEORIES)
1. Reaction between gases in magma2. Decomposition of methane to graphite
and hydrogen at 600oC temperatures3. Reaction between CO2, CH4, H2O at
elevated temperatures in vapours4. Radiolysis of water 5. Cataclasis of silicates under stress 6. Hydrolysis by ferrous minerals in mafic
rocks
HYDROGEN GENERATION
THE SUBSURFACE ENVIRONMENT
MacrohabitatsAncient salt depositsCavesCritical Zone Marine sediments
MicrohabitatsCommunity StructureDistribution
SUBSURFACE ENVIRONMENTS
NutrientsOxygenpHPorosityRadiation
SalinityTemperatureTectonic activityWater
ENVIRONMENTAL CONDITIONS
Prokaryotes Bacteria Archaea
Eukaryotes Fungi Algae Protozoa
VirusesConstraints: microhabitat size and water
availability
CRITICAL ZONE
Surface MR = 10 -3 to 10 -1 g C/g cell C/hourSubsurface MR = 10 -7 to 10 -5 g C/g cell C/hour
SURFACE VS. SUBSURFACEMETABOLIC RATES
Photosynthesis-independentIndigenous or imported nutrients?
Sedimentary C H2 or methane (earth’s centre)
Oxidation of organic matter coupled to electron acceptors at slower ratesMean generation time = thousands of years!
METABOLISM
TERMINAL ELECTRON ACCEPTING PROCESSES (TEAP)
TEAPS (CONT’D)
Quantitatively measures:Abundance and distributionViable biomassCommunity compositionNutritional/physiological status
PLFA = viable; DGFA = non-viable
PHOSPHOLIPID FATTY ACID (PLFA) ANALYSIS
Are subsurface bacteria less resistant to UV radiation than surface bacteria?
Surprisingly comparable UV resistance as surface microbes
Critical conservation of DNA repair pathwaysChemical insults e.g. oxygen radicals
Physiological characteristicsPigmentation, cell wall thickness
ADAPTATIONS
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 long-term
Ribosomes and cell walls detected by FISH
ARE THEY ASLEEP? (BACTERIAL DORMANCY)
Sporadic growth
Slow growth rates
Periods of dormancy
Adaptation to habitat variability
ADAPTIVE METABOLIC STRATEGIES
SAMPLING AND ANALYTICAL
TECHNIQUES
Main method of extraction: DrillingSamples must be properly extracted to
avoid contaminantsMajor contaminant is drilling fluidSterility of core samples confirmed by
testing core samples for the presence of drilling fluid
EXTRACTION AND SAMPLING
ANALYTICAL TECHNIQUES
Cultivation DependentDirect count of
OrganismsGrowing of the
MicroorganismBiochemical Activity
Cultivation Independent (Molecular)• RNA analysis• Denaturing gel
electrophoreses• RFLP• FISH analysis• More….
CG content analysis DNA homologyRNA analysis
- probes - 16S rRNA - in situ Hybridization
Genomics, Metagenomics and Proteomics
Problems and Complications
MOLECULAR TECHNIQUES
STUDIES OF THE SUBSURFACE
Under Construction…
NEW DNA EXTRACTION METHOD
Archaea dominate the subsurfaceLower permeability of cell membraneEnergized membrane, lower energy costs
Mediate methane production and consumption
SUBSURFACE ARCHAEA
Ancient Archaeal GroupDeep-Sea Hydrothermal
Vent Euryarchaeota l Group 6
Marine Benthic Group BMarine Benthic Groups A&DMarine Group I ArchaeaMarine Hydrothermal Vent
GroupMiscellaneous
Crenarchaeotic GroupSouth African Goldmine
Euryarchaeotal GroupTerrestrial Miscellaneous
Euryarchaeotal Group
SUBSURFACE ARCHAEA (CONT’D)
Isotope-labelled cells did not hybridize with Archaeal organismsMethodological difficulty of the techniqueUncharacterized phylogenetic diversity
Primer mismatchesUnequal distribution between the groups
Inaccurate representation of the Archaeal groups
PROBLEMS WITH CHARACTERIZATION
Suggests unsampled subsurface diversity!
PROBLEMS WITH CHARACTERIZATION (CONT’D)
New primer combinations/designsMany uncharacterized ArchaeaBetter integration of phylogenetic and
biogeochemical observations
FUTURE IMPLICATIONS
CHALLENGES OF STUDYING THE
SUBSURFACE
High possibility of contaminationStudy of subsurface microorganisms survival rate to UV radiation and hydrogen peroxide
Inaccuracies in quantificationClassical culturing techniques unable to describe the total microbial community
In situ and in laboratory disparities
CHALLENGES OF STUDYING THE SUBSURFACE
Clean drilling equipmentAseptic containment of samplesTracers in drilling fluidSample surrounding environment Immediate on-site analysis
PREVENTION OF CONTAMINATION
FUTURE DIRECTIONS
Exploit microbial metabolism Radioactive wastes in the subsurfaceEx. Pseudomonas spp. in Antarctica used to metabolize xenobiotic compounds
BIOREMEDIATION
No method for proper storage/disposalUse 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.
NUCLEAR WASTE DISPOSAL
Limited growth and survival conditions Understanding habitability of deep
subsurface can be extrapolated to habitability of other planets and Astrobiology
EXTREMOPHILES AND ASTROBIOLOGY
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)
WHY CARE ABOUT THE SUBSURFACE?
Definition of “deep subsurface”TheoriesEnvironment, Metabolism, and
AdaptationsMolecular techniques > CultivationArchaea dominate the subsurfaceContamination is a major issueSubsurface MOs have a wide range of
uses!
SUMMARY
?QUESTIONS?