The study of deep life

Isabelle Daniel

Coll. Aude Picard & Philippe Oger

Depth- & pressure range for life

Biosphere limited to …Earth ‘superficial’ layers

0 - n x0.1 GPa or n GPa?

Constraints on the extent of deep life:• Stability of biomolecules • Stability of cell units• Growth• Metabolic activities of living organisms• Nutrient source• Etc…

Not to scale

0.1 MPa

365 GPa

High-pressure environments hostthe ‘unseen majority’

• No sunlight• High hydrostatic pressure• Microorganisms- heterotrophs, dependent on nutrients from the overlying productive ocean - chemoautotrophs

dP=gdz

Mid-oceanic ridge

Hydrothermal vents

DEEP SEA

Cold deep sea

Oceans10 MPa/km

Continental crust27-30 MPa/km

Oceanic crust27-32 MPa/km

Sediments15-25 MPa/km

SUBSURFACE

SUBSEAFLOOR

Whitman et al., 1998, PNAS, 95, 6578

Subsurface

Temperature gradient:10-30°C/km

Subsurface biosphere

• Estimated biomass 0.25-2.5 1030 cells(Whitman, et al., 1998)

• Diversity of environments:MinesSandstonesGranitic aquiferes …

Not much is known…Life probably limited by temperature increase…

Hydrothermal vents

2°C

350°C

1200°COceanic crust

FeOOH, MnO2, FexSy, Mn2+, Fe2+…

3He, H2, CH4, H2S…

Riftia pachyptilia

Black smoker

Cold deep sea

Recycling of OM

≈ 2°C

Deep marine biosphereEstimated population (Witman et al., 1998)

• Heterotrophs - above 200 m, 3.6 1028 cells - below 200 m, 8.2 1028 cells• Autotrophs

2 1027 cells• TOTAL 1.2 1029 cells

55% of all the prokaryotes found in aquatic habitats

Subseafloor

Temperature gradient: 7-15°C/km

Subseafloor biosphere

• Estimated population : 3.5 1030 cells (Whitman, et al., 1998)

• Alive! >1.3 1029 cells (Schippers et al., 2005)

• Cell density decreases with depth… but at chemical interfaces (Parkes et al., 2005)

• Sulfate reduction and methanogenesis are the dominant energetic metabolisms

Living bacteria from the the sub-seafloorP ≈ 56 MPa

Site 1230, 143 mbsf

Bacteria detected by CARD-FISH in a sediment core fromthe Peru trench (5 086 m)

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From Schippers et al., 2005, Nature,433, 861

Depth profile of totalprokaryotes (),

bacteria (x),Archea ()

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Depth profile ofprokaryotes AODC ()

bacteria CARD-FISH(),bacteria Q-PCR (x),

How deep does this extend?

How active are the cells?

How deep does this extend?

• Seafloor surface: 11 km depth, 110 MPain the Mariana Trench- Colwellia MT41 : 103 MPa, 281 K- Shewanella benthica DB172F : 70 MPa, 283 K- Moritella yayanosi DB21MT-5: 80 MPa, 283 K

• Subseafloor: 70 - 80 MPa, data down to 500 m in the sediment columnex : methane production in the Japan seaex : in the Peru trench,…

At greater depth, only extrapolation available…(Parkes et al., 1994, Nature, 371,410)

… a sediment core in the Mariana trench?

in D’Hondt et al., 2002, Science , 295, 2067

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How active are the cells?

• The cell densities decrease by ca. 7fold for each 10fold increase in depth or age of the sediments

• The reactivity of organic matter decreases 10fold for each 10fold increase in age

• Thus, the availability of organic matter by cell is extremely low…

• The ‘deepest’ biosphere is practically non growing

• This challenges our understanding of the minimum energy requirements for life, for the stabilisation of the biochemical machinery

in Jørgensen & Boetius, 2007, Nature Reviews Microbiology , 5, 770)

Energy sources for the ‘starving majority’

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e- donor / e- acceptor

OM, H2, CH4, or…* / O2

* H2S, S2O32-, Fe2+, Mn2+

OM or H2 / NO3-

OM or H2 / Mn4+

OM or H2 / Fe3+

OM or H2 / SO42-

OM or H2 / CO2

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Idealized pore water and solid phase profiles based on the sucessive utilization of terminal electron acceptor during the decomposition of marine sedimentary OM (in Konhnauser, 2007)

Jørgensen & D’Hondt, 2006, Science,314, 932

Evolution of energetic metabolismas a function of pressure

Our VERY SIMPLE model :

alcoholic fermentationby the yeast Saccharomyces cerevisiaeA piezosensitive Eukaryote

Our method :

Raman spectroscopyin diamond anvil cell (DAC)

0.1 MPa• Optimal growth pressure

20 - 50 MPa• arrest of cell cycle

40-60 MPa• Internal acidification• Induction of stress transcriptional factors

70-200 MPa• Induction of stress transcriptional profile

220 MPa• Death

Reviews:Abe F., Cell. Mol. Biol., 2004Fernandes, et al., 2008

50 MPa

Eth

an

ol

Eth

an

ol

Eth

an

ol

In situ monitoring of alcoholic fermentation

First order kinetic reaction[Eth] = 2[Glc](1-e-kt)

[Glc] = 0.15 mol/l glucose

[Eth]f = 0.27 mol/l

k = 0.152(10) h-1

Fermentation of S. cerevisiae in the DAC, as a function of pressure

Observations : ambient to 10 MPa

• reaction twice faster• yield almost at the theoretical

limit

0

0.05

0.1

0.15

0.2

0.25

0.3

0 5 10 15 20 25 30

0.1 MPa

10 MPa

Time (h)

5 MPa

yield 95%k = 0.152(10) h-1

yield 98%k = 0.345(22) h-1

Picard, A. et al., 2007. Extremophiles

Interpretation

• enhanced uptake of glucose• enhanced activity of one/several enzymes of the glycolysis and/or fermentation pathways.• no measurable lag phase excludes pressure-induced increase in protein synthesis.• more efficient expellation ethanol from the cell under pressure, due to an increase of passive diffusion.

Fermentation of S. cerevisiae in the DAC, as a function of pressure

Observations : above 10 MPa,

• yield decreases• reaction rate almost constant

At 40 MPa, yield of 68% similar to Abe & Horikoshi (1997)

Alcoholic fermentation stops between 65 and 100 MPa

0

0.05

0.1

0.15

0.2

0.25

0.3

0 5 10 15 20 25 30

0.1 MPa

30 MPa

Time (h)

40 MPa

20 MPa

55 MPa

65 MPa

100 MPa

yield 95%k = 0.152(10) h-1

yield 28%k = 0140(21) h-1

Picard, A. et al., 2007. Extremophiles

Fermentation of S. cerevisiae at high pressure, conclusions

20-87 MPa Inhibited steps- no massive cell deathonly -1 log after 24 hours at 70 MPa- Progressive inhibition of enzymes?

0-10 MPaActivated steps- Increased uptake of glucose?- Activation of glycolysis or fermentation pathways enzymes?- Enhanced excretion of ethanol?

37 MPa higher than - the predicted value (Abe et al.,2004)- the pressure limit for growth

Pmax éthanol = 877 MPa

Uncoupling between the limits of growth and energetic metabolism

0

0,05

0,1

0,15

0,2

0,25

0,3

0 20 40 60 80 100

Eth

ano

l co

nce

ntr

atio

n (

mo

l/l)

Pressure (MPa)

Evolution of energetic metabolismas a function of pressure

model :

Metal respirationbyShewanella oneidensis MR-1

method :

X ray Absorption Near Edge Spectroscopy at high pressureDAC,Autoclave,Pressure vesselssee Oger et al., Spectrochimica Acta, 2008

- same phylum as major cultivated piezophilic bacteria -Proteobacteria,- autochtonous in suboxic or anoxic sediments

Optimal temperature 30°CPiezosensitive

Respiration by Shewanella oneidensis MR-1

Electron acceptors

Electron donors

Lactate

Amino acids(Yeast extract)

Pyruvate

Formate

H2

InorganicsO2

NO3-, NO2

-

S0, S2O32-

V(V)Cr(VI) Mn(IV) Fe(III)Se(IV)

OrganicsFumarateTMAODMSO

Electron binding energies at K edge:

S (2.47 keV)V (5.47 keV)Cr (5.90 keV)Mn (6.54 keV)Fe (7.11 keV)Se (12.65 keV)

Accessible compounds by X-ray spectrocopy

Selenite respiration by MR-1

120 MPa/30°C

Lactate Yeast extract

SeO32-

AcetatePyruvate…

Red elemental selenium produced under high hydrostatic pressure

Selenite respiration by MR-1as a function of pressure

0-45 MPaComplete reduction of selenite

45-150 MPaDecrease of reaction yield

0-150 MPaDecrease of reaction rate

109 cells/ml

0

0.2

0.4

0.6

0.8

1

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 20 40 60 80 100 120 140 160

Pressure (MPa)

Selenite respiration by Shewanella oneidensis MR-1as a function of pressure

In situ, DACIn situ, autoclaveEx situ, pressure vessel

Se(IV) reduction,Pmax = 155 5 MPa

65 - 155 MPa Decrease of efficiency of respiratory chain?Inactivation of ATPase?

0 - 65 MPaEfficient respiratory chain

105 MPa higher thanpressure limit for growth

Uncoupling between the limits of growth and energetic metabolism

Concluding remarks

Uncoupling between growth and metabolism

An active microorganism is not necessarily a growing microorganism Contribution of microorganisms to deep biogeochemical cycles maybe significantly underestimated

Pressure limit

Growth Energetic metabolism

Saccharomyces cerevisiae

50 MPa 87 MPa

Shewanella oneidensis MR-1

50 MPa 159 MPa

How far in pressure are the limits of activity of piezophile microorganisms?