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 Studies 5. Challenges 6. Why Care about the Subsurface?  Future directions SEMINAR OUTLINE

WHAT IS THE DEEP SUBSURFACE?

 Varies according to different disciplines  Arbitrary numbers  Microbiological definition  Hydrologic 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 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 ENVIRONMENTS FOR INTRATERRESTRIAL LIFE

 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? WHAT IS GOING ON DOWN THERE? (THE THEORIES)

1.Reaction between gases in magma 2.Decomposition of methane to graphite and hydrogen at 600 o C temperatures 3.Reaction between CO 2, CH 4, H 2 O at elevated temperatures in vapours 4.Radiolysis of water 5.Cataclasis of silicates under stress 6.Hydrolysis by ferrous minerals in mafic rocks HYDROGEN GENERATION

THE SUBSURFACE ENVIRONMENT

Macrohabitats  Ancient salt deposits  Caves  Critical Zone  Marine sediments Microhabitats  Community Structure  Distribution SUBSURFACE ENVIRONMENTS

 Nutrients  Oxygen  pH  Porosity  Radiation  Salinity  Temperature  Tectonic activity  Water ENVIRONMENTAL CONDITIONS

 Prokaryotes  Bacteria  Archaea  Eukaryotes  Fungi  Algae  Protozoa  Viruses  Constraints: microhabitat size and water availability CRITICAL ZONE

Surface MR = to g C/g cell C/hour Subsurface MR = to g C/g cell C/hour SURFACE VS. SUBSURFACE METABOLIC RATES

 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! METABOLISM

TERMINAL ELECTRON ACCEPTING PROCESSES (TEAP)

TEAPS (CONT’D)

Quantitatively measures:  Abundance and distribution  Viable biomass  Community composition  Nutritional/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 pathways  Chemical insults e.g. oxygen radicals  Physiological characteristics  Pigmentation, 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: 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 EXTRACTION AND SAMPLING

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….

 CG content analysis  DNA homology  RNA 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 subsurface  Lower permeability of cell membrane  Energized membrane, lower energy costs  Mediate methane production and consumption SUBSURFACE ARCHAEA

 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 SUBSURFACE ARCHAEA (CONT’D)

 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

 Suggests unsampled subsurface diversity! PROBLEMS WITH CHARACTERIZATION (CONT’D)

 New primer combinations/designs  Many uncharacterized Archaea  Better integration of phylogenetic and biogeochemical observations FUTURE IMPLICATIONS

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 CHALLENGES OF STUDYING THE SUBSURFACE

 Clean drilling equipment  Aseptic containment of samples  Tracers in drilling fluid  Sample surrounding environment  Immediate on-site analysis PREVENTION OF CONTAMINATION

FUTURE DIRECTIONS

 Exploit microbial metabolism  Radioactive wastes in the subsurface  Ex. Pseudomonas spp. in Antarctica used to metabolize xenobiotic compounds BIOREMEDIATION

 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. 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”  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! SUMMARY

? QUESTIONS?