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?