Hydrothermal Vent Research Linked to the Carnegie NAI Team

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Hydrothermal Vent Research Linked to the Carnegie
NAI Team

• John A. Baross
• Astrobiology Graduate Students
• Matt Schrenk - rock hosted microbial habitats,
  biofilms
• Julie Huber - subseafloor microbial ecology,
  primary producers
• Billy Brazelton - Lost City microbial ecology,
  methane metabolism
• Mausmi Mehta - N fixation at vents
• Jon Kaye (Ph.D. 2003) - salt and pressure effects

Sampling blue mat at Village Vent - Axial Seamount
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Key themes in hydrothermal vent research linked
to Carnegie NAI

• Parallel environments - subseafloor and
  rock-hosted ecosystems
• Limits of life
• Novel metabolic pathways and evolutionary
  implications
• Physiological adaptations - biofilms, exploiting
  nutrients from minerals, consortial interactions
• Biosignatures and life detection

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TOPICS

• OVERVIEW OF WORK AT VENTS - Rock Hosted Microbial
  Ecosystems - magma- and peridotite-hosted
  hydrothermal systems, subseafloor crustal
  habitats etc
• NAI TARGETED RESEARCH - Limits of Life, Biofilms,
  Novel Primary Producers at Vents related to
  early evolution of metabolic pathways, etc
• Collaborative Research Ideas

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Magma-Hosted Hydrothermal Systems

• Microbial habitats
• Diffuse flow vents - windows into the subseafloor
• Smoker fluids and sulfides
• Plumes
• Microbial mats
• Animals
• Flanks- subseafloor

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Cross Section Through a Cut-Face of Finn Showing
Mineralogical Composition and Fluid Flow Patterns
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Microbial Biomass vs. Mineralogy within a Black
Smoker Sulfide Chimney
Cryo-SEM
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DAPI and FISH-direct count analysis of microbial
populations within FINN (Cells/g dry wt sulfide
X105)
Percentage of probed cells/DAPI-stained total
population (Schrenk et al., 2003)
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Faulty Towers complex in the Mothra Hydrothermal
Field
Finn
Archaeal 16S r RNA phylogenetic diversity data
from the Finn sulfide structure (gt300C fluids)
Finn
Schrenk et al., 2003
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Novel Isolates from Active Sulfide Structures
Nannoarchaeum equitans and Ignicoccus spp
Hyperthermophilic Fe III reducer
from Kashefi and Lovley, 2003
From Huber et al., 2003
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Peridotite Hosted Hydrothermal Systems - Lost
City example

• Characteristics

Ultramafic oceanic crust Fluid flow sustained by
sub-seafloor serpentinization reactions High pH,
moderate temperature, volatile-rich fluids Tall
carbonate chimneys (60 m) and estimated to be
gt30,000 years old
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SOURCE REACTIONS FOR HYDROGEN

• (MgFe)2SiO4 H2O ?
• olivine
• Mg3Si2O5 (OH)4 Fe3O4 H2
• serpentine magnetite
• Fe2SiO4 (fayalite) is the active H2 producing
  compound of olivine

Hydrogen in combination with Ni and Fe-bearing
minerals can result In the synthesis of methane
and other low MW C-compounds
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Carbonate Chimney Ecosystems

• Highly porous chimneys with numerous flanges
• Venting up to 90?C, pH 11 fluids
• -high in volatiles
• -low in metals, H2S
• Some degree of seawater influence

Kelley, Nature (2001)
flange
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55C fluids
40µm
20µm
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Low archaeal diversity in active carbonate
chimney samples from Lost City

a. F420 autofluorescence of a biofilm b. Biofilm
cells encased in EPS c. F420 autofluorescence of
methane-metabolizing Archaea d. FISH
photomicrograph confirming that the
F420-fluorescent cells are Archaea
16S rRNA tree from Schrenk and Brazelton,
unpublished
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Detection of mcrA genes in Lost City carbonate
samples indicate the presence of a distinct
Methanosarcinales phylotype and a group related
to AMME-1
Methanogenic pathway
CO2
?
Coenzyme F420
Many steps
?
?
CH4-S-CoM
Methyl-coenzyme M reductase
?
CH4
Brazelton et al., unpublished
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Microbial diversity in Lost City carbonates and
magma-hosted sulfide chimneys a comparison

• Lost City carbonates
• Microbial community diversity varies with
  location in carbonates
• Microbial communities at pH gt10 and Temperatures
  80-90C
• One archaeal phylotype dominates
• Low bacterial diversity dominated by groups seen
  in magma-hosted vent systems
• Biofilms dominate

• Sulfide chimneys
• Microbial community diversity varies with
  location in sulfide
• Evidence of intact microbes at temperatures
  gt150C
• Extremely high diversity of uncultured archaea
• Bacteria dominate on outer wall and archaea
  dominate inner sections of sulfides
• Biofilms dominate

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Biofilms at Vents

• Microscopic and macroscopic observation on rocks
  and sulfides
• Most thermophilic and hyperthermophilic bacteria
  and archaea isolated from subseafloor and
  sulfides form biofilms
• The primary producers isolated from subseafloor
  environments form biofilms

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GR1
1 µm
10 µm
1 µm
GR1 cultured with FeOOH showing attachment (F)
and biofilm formation (C, D) TEM showing
flagella (E)
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High-temperature, high-pressure microcosm
experiments

• Attempt to mimic natural ecosystems
• -T/C gradients
• -pressure
• Use conditions which favor the formation of
  biofilms
• Use model organisms representing key phylogenetic
  and physiological groups found at vents

McCollom and Shock, GCA (1997)
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Roles of biofilms in the vent environment?

• Utilization of surface-bound resources
• Stability in a dynamic system
• Consortial interactions
• Response to environmental stress

S2O3
Fe3
hydrothermal fluids
seawater
S
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Biofilms responses to environmental stress

• Key Questions
• How do attached communities respond to
  environmental stresses?
• How do they compare physiologically to organisms
  grown under optimal conditions?
• What are the limits to cell viability?
• What bio-molecules are preserved under
  sub-optimal conditions?
• Can we use the concept of reverse chemical
  evolution to determine the bio-molecular upper
  temperature limits to life?

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Biofilm formation in laboratory microcosms

• Complex microbial biofilms have been formed in
  laboratory microcosms
• After long periods of incubation, a majority of
  the active cell population (gt10X the planktonic)
  is attached to surfaces
• The most prolific films develop when the cells
  are in stationary growth phase.

Pyrococcus str. ES-4, 90 C
Cells ml-1 fluid
_at_1000x
_at_200x
of surface coverage
time (h)
Schrenk, et al., unpublished
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Pyrococcus str. Es-4
A range of physiologies evolve within the biofilm
communities, even though they originate from
mono-clonal cultures
10 µm
Schrenk, et al., unpublished
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Microbial responses to perturbations
25 µm
50 µm
Pyrococcus str. ES-4 stained following several
cycles of tidal flushing

• Environmental perturbations such as oxygen
  stress, temperature stress, starvation, and
  changes in nutrient supply affect the composition
  and magnitude of the biofilms

Schrenk, et al., unpublished
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Upper temperature limits to life?

• Cell-like objects which fulfill life-detection
  criteria at temperatures well above 121C
• Putative biomass is aggregated around
  precipitated mineral particles
• Follow-on research is investigating these
  structures using several independent methods

200250C
SYTO11
40 µm
200250C
SYTO11
20 µm
Schrenk, et al., unpublished
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Multi-faceted analytical approach

• Microscopy
• Characterize microbial biochemistry and
  physiology
• -Dyes targeting key biomolecules
• -Merge with micro-autoradiography to follow
  metabolic pathways
• NMR (w/ Cody?)
• Analyze the macro-molecular composition of high
  temperature biofilms
• Follow compositional changes within different
  growth regimes
• Microarrays (w/ Steele?)
• Biopolymer-targeted antibody microarrays

DAPI
10 µm
ConA
Pyrococcus str. ES-4 stained with DAPI and
Concanavalin A (EPS)
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The Subseafloor Biosphere-An active microbial
ecosystem that it independent of photosynthesis?

• What phylogenetic groups of microorganisms are
  the primary producers and what metabolic
  pathways do they use? (CO2 fixation,
  heterotrophy, etc)
• What are the sources and kinds of electron
  acceptors used by subseafloor microorganisms?
• What are sources of N, P and organic matter used
  by subsurface microorganisms?

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Primary Producers in the Subseafloor

• CO2 Fixation
• 1. Calvin-Benson cycle aerobic sulfur
  oxidizing bacteria
• 2. Reductive acetyl-CoA pathway methanogens
  Desulfobacterium sp
• 3. Reductive TCA cycle Aquifacles group
  ?-proteobacteria, Thermoproteaceae and other
  Crenarchaeota
• 4. 3-hydroxypropionate pathway Metallosphaera
  sedula
• 5. Unknown and probably novel Ignicoccus spp,
  Pyrodictiaceae (Calvin cycle?) for example

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Multi-faceted analytical approach

• Microscopy
• Characterize microbial biochemistry and
  physiology
• -Dyes targeting key biomolecules
• -Merge with micro-autoradiography to follow
  metabolic pathways
• NMR (w/ Cody?)
• Analyze the macro-molecular composition of high
  temperature biofilms
• Follow compositional changes within different
  growth regimes
• Microarrays (w/ Steele?)
• Biopolymer-targeted antibody microarrays

DAPI
10 µm
ConA
Pyrococcus str. ES-4 stained with DAPI and
Concanavalin A (EPS)
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The Subseafloor Biosphere-An active microbial
ecosystem that it independent of photosynthesis?

• What phylogenetic groups of microorganisms are
  the primary producers and what metabolic
  pathways do they use? (CO2 fixation,
  heterotrophy, etc)
• What are the sources and kinds of electron
  acceptors used by subseafloor microorganisms?
• What are sources of N, P and organic matter used
  by subsurface microorganisms?

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Primary Producers in the Subseafloor

• CO2 Fixation
• 1. Calvin-Benson cycle aerobic sulfur
  oxidizing bacteria
• 2. Reductive acetyl-CoA pathway methanogens
  Desulfobacterium sp
• 3. Reductive TCA cycle Aquifacles group
  ?-proteobacteria, Thermoproteaceae and other
  Crenarchaeota
• 4. 3-hydroxypropionate pathway Metallosphaera
  sedula
• 5. Unknown and probably novel Ignicoccus spp,
  Pyrodictiaceae (Calvin cycle?) for example

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• Ax99-59 isolated from Axial Volcano
• Strict anaerobe
• Thermophilic
• CO2 is carbon source
• H2 as energy source
• Reduces sulfur species
• 32 min doubling time under
• optimal conditions
• GC ratio if 40
• New genus in the
• Aquifacales

Scanning electron micrograph of Ax99-59. Under
most culturing conditions this organism produce
copious amount of exo- polysaccharide, which may
be involved in Biofilm formation. Scale bar is 1
µm
Huber, unpublished
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• Isolated from 24 ºC diffuse fluid vent
• Strict anaerobe
• Electron Acceptors
• Polysulfide, Sº, Sulfite, and Thiosulfate
• Electron Donors
• H2, dimethylamine, methanol, formate and
  acetate
• Carbon Sources
• CO2 only
• Doubling Time
• 32 minutes!

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How is Ax99-59 fixing CO2?

• 4 known paths of CO2 fixation in prokaryotes-
  certainly other novel pathways, i.e. Igniococcus
  spp.
• Reductive Citric Acid Cycle (rTCA)- potentially
  most ancient autotrophic CO2 fixation pathway
• rTCA is important at vents
• Genes detected in vent isolates Aquifex spp,
  e-proteobacteria and hyperthermophilic
  Crenarchaeota
• Genes detected and expressed in vent
  environmental samples

aclB
aclB ATP citrate lyase
oorA oxoglutarateferredoxin
oxidoreductase
oorA
Wächtershäuser, 1990 Beh et al. 1993 Cody et
al. 2001 Hügler et al. 2003 Campbell et al.
2003, 2004
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ATP citrate lyase (aclB)
UM
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Ancient Metabolic Pathways and the Unity of
Metabolism

• Reductive TCA Pathway - reduce CO2 - dominant in
  magma-hosted vent environments
• Reductive AcetylCoA Pathway - reduce CO2 to CH4
  and in reverse oxidize CH4 to (CH2O)n - Lost city
  primary producer
• Oxidation of C1 and C2 compounds with Fe(III) or
  S - dominant in magma-hosted vent environments
• At least two unknown CO2-fixing pathways in
  Archaea - all from vent environments

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Some ideas for collaborative research projects

• How many novel C1(CO2, CO, CH4, etc) utilizing
  metabolic pathways exist?
• Do deep-sea vents retain organisms which can
  utilize non-enzymatically catalyzed mineral
  surface reactions as part of their metabolism?
• Are there vent microorganisms that are dependent
  on organic compounds synthesized abiotically by
  mineral catalyzed reactions?
• Assuming a unity of metabolism on other planets
  and moons given common geochemical and
  geophysical properties, can we identify key
  biosignatures for metabolic pathways?