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

New Opposed Anvil CellNew Opposed Anvil CellNew Opposed Anvil CellNew Opposed Anvil Cell

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.

Ice VI and VIIIce VI and VII

2. Volumes of Reaction, ΔV2. Volumes of Reaction, ΔV

Pressure may suppress reactions if ΔV > 0.

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

ATPATP POPO44

+ ADP+ ADP

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.

With thanks to:

NASA Astrobiology Institute National Science Foundation

Carnegie Institution of Washington