Can High Pressures Enhance The Long-Term Survival of Microorganisms?
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Can High Pressures Enhance Can High Pressures Enhance The Long-Term SurvivalThe Long-Term Survival
of Microorganisms?of Microorganisms?
Can High Pressures Enhance Can High Pressures Enhance The Long-Term SurvivalThe Long-Term Survival
of Microorganisms?of Microorganisms?
Robert M. Hazen & Matt SchrenkRobert M. Hazen & Matt SchrenkCarnegie Institution & NASA Astrobiology InstituteCarnegie Institution & NASA Astrobiology Institute
Pardee Keynote Symposium: Evidence for Long-Term Survival of Pardee Keynote Symposium: Evidence for Long-Term Survival of Microorganisms and Preservation of DNAMicroorganisms and Preservation of DNA
Philadelphia – October 24, 2006Philadelphia – October 24, 2006
ObjectivesObjectives ObjectivesObjectives
I. Review the nature and distribution of life Review the nature and distribution of life at high pressures.at high pressures.
II. Examine the extreme pressure limits of II. Examine the extreme pressure limits of cellular life.cellular life.
III. Compare experimental approaches to III. Compare experimental approaches to study microbial behavior at high P and T.study microbial behavior at high P and T.
IV. Explore how biomolecules and their IV. Explore how biomolecules and their reactions might change at high pressure?reactions might change at high pressure?
I. High-Pressure LifeI. High-Pressure LifeI. High-Pressure LifeI. High-Pressure Life
HMS Challenger – 1870s20 MPa (~200 atmospheres)
High-Pressure LifeHigh-Pressure LifeHigh-Pressure LifeHigh-Pressure Life
Deep-Sea HydrothermalDeep-Sea HydrothermalVents – 1977Vents – 1977
High-Pressure LifeHigh-Pressure Life
Discoveries of Microbial Life in Crustal Rocks – 1990sDiscoveries of Microbial Life in Crustal Rocks – 1990s
P ~ 100 MPaP ~ 100 MPa
Witwatersrand Deep Witwatersrand Deep Microbiology ProjectMicrobiology Project
Energy from radiolytic Energy from radiolytic cleavage of water. cleavage of water.
Lin, Onstott et al. (2006) Lin, Onstott et al. (2006) ScienceScience
Thomas Gold’s Hypothesis:Thomas Gold’s Hypothesis:Organic Synthesis in the MantleOrganic Synthesis in the Mantle
Thomas Gold’s Hypothesis:Thomas Gold’s Hypothesis:Organic Synthesis in the MantleOrganic Synthesis in the Mantle
Thomas Gold (1999) Thomas Gold (1999) NY:Springer-Verlag.NY:Springer-Verlag.
>300 MPa>300 MPa
Life at High Pressureson other Worlds?
Life at High Pressureson other Worlds?
Deep, wet environments may exist on Mars, Europa, etc.Deep, wet environments may exist on Mars, Europa, etc.
I. What is the Nature I. What is the Nature and Distribution of Life and Distribution of Life
at High-Pressure?at High-Pressure?
I. What is the Nature I. What is the Nature and Distribution of Life and Distribution of Life
at High-Pressure?at High-Pressure?
Deep life, especially Deep life, especially microbial life, is abundant microbial life, is abundant
throughout the upper crust . throughout the upper crust .
II. What are the Extreme II. What are the Extreme Pressure Limits of Life?Pressure Limits of Life? II. What are the Extreme II. What are the Extreme Pressure Limits of Life?Pressure Limits of Life?
How deep might microbes How deep might microbes exist in Earth’s crust?exist in Earth’s crust?
Pressure Can Enhance T StabilityPressure Can Enhance T StabilityPressure Can Enhance T StabilityPressure Can Enhance T Stability
[Margosch et al. (2006) Appl. Environ. Microbiology 72, 3476-3481.]
[Pledger et al. (1994) FEMS Microbiology Ecology 14, 233-242.]
Life’s Extreme Pressure LimitsLife’s Extreme Pressure LimitsLife’s Extreme Pressure LimitsLife’s Extreme Pressure Limits
Fast, cold slabs have regions of P > 2 GPa & T < 150°C [Stein & Stein (1996) AGU Monograph 96]
60 km60 km
Hydrothermal Hydrothermal
Opposed-Anvil CellOpposed-Anvil Cell
Pressure Limits of LifePressure Limits of LifePressure Limits of LifePressure Limits of Life
Pressure Limits of LifePressure Limits of LifePressure Limits of LifePressure Limits of Life
Microbes and their environment can be probed optically at
pressure.
[Sharma et al. (2002) Science 295, 1514-1516]
E. coli
1 atm
1.4 GPa1 hour
1.4 GPa>30 hours
Pressure Limits of LifePressure Limits of LifePressure Limits of LifePressure Limits of Life
Ice VI forms at P > 1.0 GPa & 25°C. Microbes
persist at triple junctions
[Sharma et al. (2002) Science 295, 1514-1516]
E. coli
1 atm
1.4 GPa1 hour
1.4 GPa>30 hours
Pressure Limits of LifePressure Limits of LifePressure Limits of LifePressure Limits of Life
Raman measurements show formate reduction
to P > 1.4 GPa.
Microbes shape their icy environment and viable microbes are
recovered after several days at 1.4 GPa & 25°C.
[Sharma et al. (2002) Science 295, 1514-1516]
E. coli
1 atm
1.4 GPa1 hour
1.4 GPa>30 hours
II. What are the Extreme II. What are the Extreme Pressure Limits of Life?Pressure Limits of Life?II. What are the Extreme II. What are the Extreme Pressure Limits of Life?Pressure Limits of Life?
Some microbes survive at Some microbes survive at P > 1.4 GPa (~14,000 atm). P > 1.4 GPa (~14,000 atm).
Temperature limits are not Temperature limits are not known, but pressure enhances known, but pressure enhances T-tolerance for some microbes. T-tolerance for some microbes.
Hydrothermal
Opposed-Anvil Cell
III. Techniques for Studying III. Techniques for Studying Microbes at High PressureMicrobes at High Pressure
III. Techniques for Studying III. Techniques for Studying Microbes at High PressureMicrobes at High Pressure
High-Pressure TechniquesHigh-Pressure TechniquesHigh-Pressure TechniquesHigh-Pressure Techniques
Gold tube reactors in hydrothermal bombsGold tube reactors in hydrothermal bombs
Flow-through ReactorFlow-through Reactor
[Jannasch et al. (1996) Appl. & Environ. Microbiology 62, 1593-1596][Jannasch et al. (1996) Appl. & Environ. Microbiology 62, 1593-1596]
High-Pressure TechniquesHigh-Pressure TechniquesHigh-Pressure TechniquesHigh-Pressure Techniques
Flow-through ReactorFlow-through Reactor
High-Pressure TechniquesHigh-Pressure TechniquesHigh-Pressure TechniquesHigh-Pressure Techniques
New Opposed Anvil CellNew Opposed Anvil CellNew Opposed Anvil CellNew Opposed Anvil Cell
“Large” volume:100 mm100 mm33 at 200 MPa at 200 MPa10 mm10 mm33 at 500 MPa at 500 MPa
Easy optical accessRapid loadingHeating optionsCryo-quenchingInexpensive
Moissanite (SiC) anvilsFour-screw designAluminum gaskets
5 cm
Studies employ ~50 pressure cellsStudies employ ~50 pressure cells
Microbe Pressure Cell ArrayMicrobe Pressure Cell ArrayMicrobe Pressure Cell ArrayMicrobe Pressure Cell Array
Redox Potential Redox Potential
Microbe Pressure Cell ArrayMicrobe Pressure Cell ArrayMicrobe Pressure Cell ArrayMicrobe Pressure Cell Array P
ressure
Pressu
re
Pressu
re P
ressure
pH pH
Microbe Pressure Cell ArrayMicrobe Pressure Cell ArrayMicrobe Pressure Cell ArrayMicrobe Pressure Cell Array P
ressure
Pressu
re
Pressu
re P
ressure
Temperature Temperature
Microbe Pressure Cell ArrayMicrobe Pressure Cell ArrayMicrobe Pressure Cell ArrayMicrobe Pressure Cell Array P
ressure
Pressu
re
Pressu
re P
ressure
Advantages of Pressure ArrayAdvantages of Pressure Array
In situIn situ observations: optical, Raman observations: optical, Raman
Significant sample volumeSignificant sample volume
Multiple variables: Multiple variables: nutrients, redox, nutrients, redox, pH, temperature, minerals, consortiapH, temperature, minerals, consortia
The anvil cell environmentThe anvil cell environment may mimic may mimic deep pockets (essentially a closed deep pockets (essentially a closed system).system).
IV. What Pressure Effects on Biomolecules Might We Study?IV. What Pressure Effects on
Biomolecules Might We Study?
1. Pressure induced phase transitions
2. Volumes of reactions
3. Activation volumes
4. Pressure effects on enzymes
5. Diffusion rates and membrane permeability
1. Pressure Induced Phase Transitions1. Pressure Induced Phase Transitions
Pressure induces phase transitions in membrane-forming lipid molecules.
2. Volumes of Reaction, ΔV2. Volumes of Reaction, ΔV
Polyphosphate cleavage (ATP ADP)
H4P2O7 + H2O 2H3PO4
ΔV ~ +15 cc/mol
for P < 1 GPa and T < 200°C
2. Volumes of Reaction, ΔV2. Volumes of Reaction, ΔV
Pressure may enhance reactions if ΔV < 0.
Methanogenesis:
CO2 + 4H2 CH4 + 2H2O
ΔV ~ -60 cc/mol
for P < 1 GPa and T < 200°C
2. Volumes of Reaction, ΔV2. Volumes of Reaction, ΔV
Methanogenesis may be enhanced by P
Methanococcus jannaschiiMethanococcus jannaschii
Methanopyrus kandleriMethanopyrus kandleri
MethanothermobacterMethanothermobacter thermoautotrophicumthermoautotrophicum
3. Activation Volumes3. Activation Volumes Activation volume is defined as the difference between the partial molar volumes of the initial state
and the transition state:
P
3. Activation Volumes3. Activation Volumes If activation volume is negative, then
reaction rates increase with P.
Bond formation; condensation
Charge concentration
FeFe2+2+ Fe3+Fe3+
3. Activation Volumes3. Activation Volumes If activation volume is positive, then
reaction rates decrease with P.
Unfolding; racemization
L-ASP D-ASP
4. Pressure Effects on Activity and Denaturation of Enzymes
4. Pressure Effects on Activity and Denaturation of Enzymes
Fructose diphosphatase (FDPase) at 150 Fructose diphosphatase (FDPase) at 150 MPa (fish)MPa (fish) Hochachka et al. (1970) Hochachka et al. (1970) Marine BiologyMarine Biology 7, 285-293. 7, 285-293.
Lactic dehydrogenase deactivation Lactic dehydrogenase deactivation (rabbits)(rabbits) Schmid et al. (1975) Schmid et al. (1975) Biophys. ChemBiophys. Chem. 3, 90-98.. 3, 90-98.
Pyrophosphatase activity (Pyrophosphatase activity (BacillusBacillus)) Morita & Mathemeier (1964) Morita & Mathemeier (1964) J. BacteriologyJ. Bacteriology 88, 88, 1667-1671.1667-1671.
Protease activity (Protease activity (MethanococcusMethanococcus)) Michels & Clark (1997) Appl. Environ. Microbiology Michels & Clark (1997) Appl. Environ. Microbiology 63, 3985-3991.63, 3985-3991.
4. Pressure Effects on Microbial Enzymes4. Pressure Effects on Microbial Enzymes4. Pressure Effects on Microbial Enzymes4. Pressure Effects on Microbial Enzymes
Use pressure cell array to:Use pressure cell array to:
Identify and analyze stress proteinsIdentify and analyze stress proteins
Measure metabolic ratesMeasure metabolic rates
Survey gene expression (using mRNA)Survey gene expression (using mRNA)
5. Diffusion rates and 5. Diffusion rates and Membrane PermeabilityMembrane Permeability5. Diffusion rates and 5. Diffusion rates and
Membrane PermeabilityMembrane PermeabilityPressure decreases diffusion rates and
membrane permeability.
Pressure increases relative diffusion rate of hydrogen
Hydrogen may be an optimal energy source for long-term survival of microbes at high P.
[Morita (2000) Microbial Ecology 38:307-320]
ConclusionsConclusions
Pressure has the potential to slow microbial Pressure has the potential to slow microbial metabolism by several mechanisms:metabolism by several mechanisms:
Positive volumes of reactions.Positive volumes of reactions.
Positive activation volumes of reactionsPositive activation volumes of reactions
Reduced enzyme activitesReduced enzyme activites
Reduced diffusion ratesReduced diffusion rates
Reduced membrane permeabilityReduced membrane permeability
More experimental studies are needed.More experimental studies are needed.