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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?