MOR hydrothermal vents
1-3: Recharge zone
• <2-150oC
• e- acceptors: O2, NO3-, SO4-2
• e- donors: Fe+, org-CSW
4-5: Reaction zone
• High To (to 400oC), fresh rock
at cracking front
• Intense water-rock reaction
• Volatile/metals leaching
• Too extreme for active life
6: Upflow zone
• Fluids at 350-400oC; low pH;
high reduced metals/volatiles
• No active life
• Some entrained fluids (w/cells)
A New Kind of
Hydrothermal Ecosystem
The Lost City Hydrothermal Field,
located 15 km from the Mid-Atlantic
Ridge, is primarily influenced by
serpentinization instead of volcanism.
Discovered in 2000 by UW
researchers and colleagues
Kelley et al. (2007)
Across Axis Morphology
Perfit and Chadwick, 1998
22
30°N Mid-Atlantic Ridge
An Oceanic Core Complex
MAR
Atlan
tis Tr
an s fo
rm Fa
ult
75 km
MAR
Atlantis
Massif
12
mm/yr
Mountains on the Seafloor
Atlantis Massif
Mountains on the Continents
Mount Rainer
21 km
25 km
17,388 feet (5,300 m)
14,435-feet tall (4,399 meter)
Origin unknown in 2000:
Active Volcano
Tectonic uplift?
Expansion from serpentinization?
Alvin:
Poster boy
Argo II towed mapping and imaging vehicle:
Actual discoverer of Lost City
Summit of 60 m-tall structure called Poseidon
Presence of carbonate chimneys strongly indicated that
serpentinization must be prevalent within the Atlantis Massif
Presence of carbonate chimneys strongly indicated that
serpentinization must be prevalent within the Atlantis Massif
Serpentinization
(hydration of olivine)
(Mg,Fe)2SiO4 + H2O + C = Mg3Si2O5(OH)4 + Mg(OH)2 +Fe3O4 + H2 +CH4 + C2-C5
(+ heat and large volume increase)
olivine + water + carbon = serpentine + high pH + energy + food
potential for supporting high-pH, non-photosynthetic ecosystems?
Olivine
Serpentine
30°N Mid-Atlantic Ridge
An Oceanic Core Complex
MAR
Atlan
tis Tr
an s fo
rm Fa
ult
75 km
MAR
Atlantis
Massif
12
mm/yr
Poseidon
Novel features:
•  Off-axis on 1.5My
old crust
•  Hosted on altered
mantle rocks
•  Circulation is driven
by geochemical
reactions
Kelley et al., 2005
•  Carbonate
precipitated from
seawater
•  No apparent
magmatic influence
Carbonate Chimneys and Flanges of Lost City
Lost City Hydrothermal Field
Warm (20-90ºC), pH 9-11 fluids
High levels of H2 (1-14 mmol/kg) and
CH4 (1-2 mmol/kg)
Dense microbial biofilms inside and
outside chimneys
Schrenk et al. (2004) Env. Microbiol.
Kelley et al. (2005) Science
SEM and TEM of chimney biofilm
archaeal biofilms
End-member Hydrothermal Environments
Sulfide systems
Carbonate systems
• Driven by the cooling of
magma/rock
• Driven by exothermic
water:rock rxns
• High T (up to 400°C)
• Moderate T (<90°C)
• Low pH fluids
• High pH fluids
• High metal concentrations
• Low metal concentrations
• Variable volatile
concentrations
• High volatile
concentrations
• Metal-sulfide chimneys
• Carbonate chimneys
Lost City Macrofauna
Very little sulfide at Lost City --> very small animals?
Macrofaunal communities are not obvious:
•  90% of fauna are <1mm in size
•  diversity is similar or greater than other sites:
13 phyla, 65 species: 58% of which are endemic
•  mostly invertebrates inhabiting pores and crevices on the exterior of
chimneys:
Snails, mussels, amphipods, polychaetes, nematodes, etc.
Lost City mussels harbor methane-oxidizing AND sulfide-oxidizing bacteria
Alien invaders
at Lost City:
Our 3am alarm
Salps: colonial tunicates
Hermaphrodites that can
reproduce asexually or sexually
Abundances can fluctuate
rapidly, carbon export can
increase greatly when their
bodies and fecal pellets sink
Temperature gradients at Lost City
Fluid source
10-40ºC, pH 8-9,
oxic
40-90ºC, pH 9-10,
anoxic
Mostly archaea
Mostly bacteria
40µm
20µm
Temperature gradients at Lost City
Fluid source
10-40ºC, pH 8-9,
oxic
40-90ºC, pH 9-10,
anoxic
Diverse bacteria
A single species biofilm
- comprises >80% of all cells
- belongs to the Methanosarcinales
group of CH4-cycling archaea
- conditions unknown to support life
Schrenk et al. (2004) Environ Microbiol
40µm
Lost City Methanosarcinales
(Background Info)
Active chimneys are dominated by a single phylotype of Archaea:
Lost City Methanosarcinales (LCMS)
–  Thick biofilms at 70-80ºC pervading carbonate chimneys
The Methanosarcinales are a group of methanogens
known for:
– Metabolic flexibility: can use CO2, acetate,
methanol, other methylated compounds
– Genomic flexibility: abundant lateral gene transfer
– Morphological diversity: life cycle of single cells
and aggregates composed of cell subtypes
Delong (2000)
Schrenk et al., 2004
Some of the anaerobic methane-oxidizing Archaea
(ANME) are also members of the Methanosarcinales
– Found in methane cold seeps (<10ºC)
– Partnered with sulfate-reducing Bacteria in syntrophic relationship
What are the metabolic and genomic characteristics of Lost City Methanosarcinales?
2005 Lost City Expedition
July 17 - August 4
On-shore PI: Debbie Kelley
On-ship PI: Bob Ballard
Onboard personnel breakdown:
10 ROV techs
6 TV personnel
3 education/public outreach
4 scientists
(1 tech, 2 grad students, 1 post-doc)
ARGUS
HERCULES
SATELLITE TRACKING ANTENNA
CONTROL VAN
2005 Expedition
Scientists cooperating 5000 miles apart, sharing
with the public across the world
UW Command Center
Shipboard Control Van
Does LC Ms produce or oxidize CH4?
13CH
13HCO
3
4
Incubate anaerobically
up to 80ºC, pH 10 for 4 - 8 days
Back in Seattle: measure
headspace with GC-MS
If 13CH4 detected, then
methanogenesis
occurred
If 13CO2 detected, then
anaerobic methane
oxidation occurred
H2-fueled CH4-cycling in Lost City chimneys
+H2 -H2
H2 stimulates:
H2 + CO2 ! CH4 and CH4 --> CO2
Therefore, both CH4 production and oxidation may occur simultaneously.
Brazelton et al. (2011) mBio
Transmission electron micrograph of chimney thin section
Diversity of archaeal lipids
1 um
- Mehay et al. (2013) Geobiology
Distribution of archaeal OTUs among samples
Abundance of each
OTU (3% distance)
normalized across
samples using
Daisy-Chopper
(from Swift, Gilbert & Field)
Complete community
shift between 100 and
1200 yrs.
ANME1 is already
present, though very
rare, at 30 yrs.
LCMS = Lost City Methanosarcinales (methanogen? or methanotroph?)
ANME-1 = anaerobic methanotroph
MGI = Marine Group I Crenarchaeota
Diversity of rare sequences within Methanosarcinacaea family
mutation rate = 0.34%
(substitution = 0.18%, indels = 0.16%)
Huse (2007) subs = 0.03%, indels = 0.13%
Two samples from the same chimney have
nearly identical profiles (95% Bray-Curtis)
100 yrs
40 yrs
30 yrs
1 um
1 um
Intracellular
membranes previously
only known in aerobic
methane-oxidizing
bacteria
Model of methanotrophic/hydrogenotrophic biofilm
CH4
CO2
CH4
CH4
H2
H2
Remember: CO2 is limiting and maybe
nonexistent
Archaea inside chimneys
HOT, high pH
CH4
CH4
CO2
H2
Bacteria outside chimneys
COLD, neutral pH
CO2
H 2S
S0
SO4
CO2
H2 consumption is explained by
sulfate reduction
- Lang et al. (2012) GCA
H2 consumption occurs where
archaea are dominant
- Mehay et al. (2013) Geobiology
Therefore, H2-fueled sulfate reduction powers methane
production and/or consumption by archaea.
Is the H2-fueled sulfate reduction mediated by the same archaea
or by bacterial partners?
Do these interpretations apply to the vast Lost City subsurface?
chimney exteriors:
aerobic sulfur-oxidizing
and methanotrophic bacteria
chimney interiors:
methanogenic /
methanotrophic archaea
ocean
electron acceptors and CO2
O2
SO42carbonate
chimneys
on seafloor
pH 11
CH4
anaerobes (e.g. Desulfotomaculum)
H2-producing? fermenting? sulfate-reducing?
subsurface serpentinization
electron donors and organic carbon
formate
H2
International Ocean Drilling Program Expedition
to the Atlantis Massif: Serpentinization and Life
http://www.eso.ecord.org/expeditions/357/357.php
Serpentinization does not require an ocean
Serpentinization
on the seafloor
chimney exteriors:
aerobic sulfur-oxidizing
and methanotrophic bacteria
chimney interiors:
methanogenic /
methanotrophic archaea
ocean
electron acceptors and CO2
Serpentinization
on continents
O2
SO42carbonate
chimneys
on seafloor
pH 11
CH4
anaerobes (e.g. Desulfotomaculum)
H2-producing? fermenting? sulfate-reducing?
subsurface serpentinization
electron donors and organic carbon
formate
H2
surface water and atmosphere
electron acceptors and CO2
O2
carbonate deposits
at surface
aerobes (e.g. Hydrogenophaga)
H2-consuming, autotrophic
anaerobes (e.g. Desulfotomaculum)
H2-producing? fermenting? sulfur-reducing?
methanogenic archaea?
subsurface serpentinization
electron donors and organic carbon
O2
pH 12
CH4
formate
H2
Locations of Serpentinite-Hosted Ecosystems
Serpentinite Spring Biogeography Project
Each of these sites hosts microbial communities fueled by serpentinization
- Does the serpentinite subsurface have a core community?
- Do the serpentinite springs show evidence of biogeographic isolation?
- Are any organisms completely independent of the surface?
Liguria Springs
(aka Voltri Massif)
Italy
Liguria Springs
(aka Voltri Massif)
Italy
Liguria Springs
(aka Voltri Massif)
Italy
Liguria Springs
(aka Voltri Massif)
Italy
Tablelands Ophiolite
Newfoundland, Canada
Tablelands Ophiolite
Newfoundland, Canada
Tablelands Ophiolite
Newfoundland, Canada
CROMO (Coast Range Ophiolite Microbial Observatory)
Northern California
CROMO
(Coast Range Ophiolite Microbial Observatory)
Northern California
CROMO (Coast Range Ophiolite Microbial Observatory)
Northern California
CSW1
.1
pH
12.5
QV1.1
pH
11.5
Serpentinite Spring Community Comparisons
Lost City
Carbonate Chimneys
(Mid-Atlantic Ridge)
Drill Well Water
(California)
Natural Springs
(Newfoundland, Italy)
Preliminary Taxonomic Survey of Serpentinite Springs
Lost City
chimneys
Tablelands Liguria
CROMO
wells
CROMO
rock cores
Taxonomic
Groups
An early subset
of the total data:
>6 million
sequences
(16S rRNA gene)
from 96 samples
Each cell
represents the
relative
abundance of one
taxonomic group
in one sample
Red = very
abundant
Low Diversity of Serpentinite Springs
Diversity
(16S rRNA,
97% OTUs)
Very low biomass:
~102 cells per mL
Why so different from
Lost City?
pH of spring water
Low Diversity of Serpentinite Springs
Italy
Beta
proteobacteria
Newfoundland
Beta
proteobacteria
California
Beta
proteobacteria
Of the few organisms found at high pH,
most are unlikely to be living in the subsurface.
Locations of Serpentinite-Hosted Ecosystems
Serpentinite Spring Biogeography Project (with Matt Schrenk, Michigan St.)
Each of these sites hosts microbial communities fueled by serpentinization
- Does the serpentinite subsurface have a core community?
- Do the serpentinite springs show evidence of biogeographic isolation?
- Are any organisms completely independent of the surface?
Tablelands Ophiolite, Gros Morne National Park,
Newfoundland, Canada
serpentinites
pool of pH 12 water
rich in H2 and CH4
!  We identify
fluid sources, insert tubing,
Tablelands
Ophiolite Spring
Serpentinite
Community
Comparisons
surface
and pump 1-50 L through 0.2 µm filters for
Newfoundland
DNA and RNA sequencing.
runoff
pH 8
•  While pumping, we monitor temperature,
pH, and Eh conditions as indicators of a
subsurface source.
mixing
zone
pH 12
•  Simultaneously, collaborators take
samples for fluid chemistry, gas
composition, organic compound analyses,
etc.
subsurface
source
pH 12
Tablelands
Ophiolite Spring
Serpentinite
Community Comparisons
surface
Newfoundland
runoff
pH 8
mixing
zone
pH 12
Hydrogenophaga
subsurface
source
pH 12
Erysipelothrix
Hydrogenophaga
Correlation Analysis of Tablelands (Newfoundland) Springs
collaboration with Penny Morrill, Memorial University of Newfoundland
7 sites x 3 time points
>100 taxa identified by fingerprinting and sequencing
Chemical variables: pH, Eh, H2, CH4, DIC, DOC
Fluid mixing model: fraction of ultrabasic fluid (fUB), Szponar et al. 2012 Icarus
Correlations between taxa and chemistry
"  Association Network
(à la Steele et al. (2011) ISME)
- Brazelton et al. (2013) Appl. Environ. Microbiol.
Correlation Analysis of Tablelands (Newfoundland) Springs
collaboration with Penny Morrill, Memorial University of Newfoundland
7 sites x 3 time points
>100 taxa identified by fingerprinting and sequencing
Chemical variables: pH, Eh, H2, CH4, DIC, DOC
Fluid mixing model: fraction of ultrabasic fluid (fUB), Szponar et al. 2012 Icarus
Hydrogenophaga
Potential microbe-chemistry and
microbe-microbe interactions are
revealed. The data are not
compressed into two dimensions.
pH
Eh
H2
Erysipelothrix
CH4
fUB
Erysipelothrix is the only taxon
with significant correlations to H2,
CH4, and fUB (fraction of ultrabasic
water)
Most likely anaerobic, possibly a
fermenter. Erysipelothrix
hydrogenases identified in
metagenomic data
(Brazelton et al., 2012. Frontiers in
Microbiol.)
H2 cycle in serpentinite springs
(tentative model based on preliminary data)
H2 cycle in serpentinite springs
(tentative model based on preliminary data)
Mixing of subsurface + surface fluids
--> Biological activity
Subsurface fluids: H2, CH4, formate, but no oxidants.
Both pH and lack of oxidants might limit biological activity.
Plenty to eat, nothing to breathe?
The Serpentinite Subsurface Remains Unexplored
Serpentinization
on the seafloor
chimney exteriors:
aerobic sulfur-oxidizing
and methanotrophic bacteria
chimney interiors:
methanogenic /
methanotrophic archaea
ocean
electron acceptors and CO2
Serpentinization
on continents
O2
SO42carbonate
chimneys
on seafloor
pH 11
CH4
anaerobes (e.g. Desulfotomaculum)
H2-producing? fermenting? sulfate-reducing?
subsurface serpentinization
electron donors and organic carbon
formate
H2
surface water and atmosphere
electron acceptors and CO2
O2
carbonate deposits
at surface
aerobes (e.g. Hydrogenophaga)
H2-consuming, autotrophic
anaerobes (e.g. Desulfotomaculum)
H2-producing? fermenting? sulfur-reducing?
methanogenic archaea?
subsurface serpentinization
electron donors and organic carbon
O2
pH 12
CH4
formate
H2
The Serpentinite Subsurface Remains Unexplored
The Big Question:
Can serpentinization support an
active ecosystem independent of
oxidants produced by the
photosynthtetic surface
biosphere?
Serpentinization
on the seafloor
chimney exteriors:
aerobic sulfur-oxidizing
and methanotrophic bacteria
chimney interiors:
methanogenic /
methanotrophic archaea
ocean
electron acceptors and CO2
Serpentinization
on continents
O2
SO42carbonate
chimneys
on seafloor
pH 11
CH4
anaerobes (e.g. Desulfotomaculum)
H2-producing? fermenting? sulfate-reducing?
subsurface serpentinization
electron donors and organic carbon
formate
H2
surface water and atmosphere
electron acceptors and CO2
O2
carbonate deposits
at surface
aerobes (e.g. Hydrogenophaga)
H2-consuming, autotrophic
anaerobes (e.g. Desulfotomaculum)
H2-producing? fermenting? sulfur-reducing?
methanogenic archaea?
subsurface serpentinization
electron donors and organic carbon
O2
pH 12
CH4
formate
H2
Mars
Serpentinization on Mars?
Evidence for ultramafics (olivine = ultramafic) on Mars:
•  composition of SNC meteorites
•  olivine-rich basalt (MGS THEMIS data)
Hamilton et al., 2005
Combine with evidence of water on Mars -> possibility of serpentinization
Serpentinization on Mars?
Evidence for ultramafics (olivine = ultramafic) on Mars:
•  composition of SNC meteorites
•  olivine-rich basalt (MGS THEMIS data)
Hamilton et al., 2005
Combine with evidence of water on Mars -> possibility of serpentinization
To maintain atmospheric CH4 at steady-state: need 8x108 kg of olivine/year
= 3.6x1017 kg over Mars history = 50 cm thick global layer of olivine
(Oze & Sharma, Geophys. Res. Lett. 2005)
Evidence for CH4 on Mars
Serpentinization
(hydration of olivine)
(Mg,Fe)2SiO4 + H2O + C = Mg3Si2O5(OH)4 + Mg(OH)2 +Fe3O4 + H2 +CH4 + C2-C5
(+ heat and large volume increase)
olivine + water + carbon = serpentine + high pH + energy + food
potential for supporting high-pH, non-photosynthetic ecosystems?
Olivine
Serpentine
Serpentinization on Mars?
H 2?
CH4?
H2S?
Challenge:
Infer activity in the subsurface
with surface-based measurements
Serpentinization on Mars? and Earth
H 2?
CH4?
H2S?
Lost City
Hydrothermal Field
Continental
Serpentinite Springs
Challenge:
Infer activity in the subsurface
with surface-based measurements
Methane as a signature of life
Lost
City
Figure from Onstott et al., Astrobiology 2006 (adapted from
Schoell, Chemical Geology 1988)
Lost City data from Kelley et al., Science 2005 and
Hypothetical CH4 cycle on Mars
data.engin.umich.edu/ PSL/research.html
Europa:Jupiter s icy Moon
Europa's Complex Geology
ridged plains
mottled terrain
Europa's
"Thick Shell"
Geology
J. Geophysical Research 106:12355-12365 (2001)
Assume a liquid water ocean
on Europa, then it is possible
that the chaos- and lenticulartype surface features could be
formed by thermal buoyant
plumes originating from
magma heating
Steady-state hydrothermal
venting could give rise to a
series of melt and freezing
events so that the overlying
ice is thinned by persistent
heating