Peering Into the Deep:
Phylogenetic and
functional diversity of
deep sea microbial
communities
Peter R. Girguis
John Loeb Associate Professor of Natural Sciences
Dept. of Organismic and Evolutionary Biology
Harvard University
[email protected]
The deep ocean
 ~ 80% of our biosphere is deep ocean
below 1,000 meters
 The average ocean depth is 4,000
meters (~2.5 miles)
 The longest mountain range on Earth is
the mid-ocean ridge system
 ~ 50,000 miles
 ~ 90% of all volcanic activity occurs in
the deep sea
The deep seafloor influences our planet’s heat, energy
budgets through hydrothermal circulation
 The ocean crustal aquifer is
the largest on Earth
 Hydrothermally-driven
circulation is rapid
 Entire volume of aquifer
expelled every 2,000-5,000
years
 Entire OCEAN every 70,000 to
200,000 years (Fisher et al, 2005)
Microbes dominate in the deep sea
 The deep sea hosts a massive
microbial community


Crustal aquifer
Sediments
 ~ 1029 microbes in ocean
sediments alone (Whitman et al. 1998)
 ~ 1015 g “Carbon” in marine
microbes (Karl and Dobbs, 1998)
 Deep sea microbes play an
major but poorly constrained
unconstrained role in marine
biogeochemical cycles
Allying microbial identity to biogeochemistry:
The grand challenge
 > 16,000 microbial genomes completed
 > 100 million known unique microbial “types”
 > 99.99% of known microbes have eluded cultivation
 Also, measuring activity at the microbial scale remains slow
How can we ally microbes to their activity?
The nature, quality and abundance of contextual
data available to ally specific microbes to
biogeochemical processes wildly varies
Tackling the “impedance mismatch”
?
 “omic-informed” experiments
 -Omics provide constraints on functional potential (NOT flux)
 Direct measurements of metabolic rate @ relevant conditions
 Co-registered microbial & geochemical studies, in lab and in situ
Hot Wired:
Interrogating
extracellular electron
transfer by thermophilic
microbes in hydrothermal
vent sulfides
The inner workings of deep sea hydrothermal vents
Prominent features of the
mid-ocean ridges, which
emit hot, chemically
reduced fluids
Buoyant fluid emerges
through crust, metals
precipitate to form
smokers
Over 200 vent fields have
been detected, far fewer
have been visited
What are the conditions like at vents?
Temperature:
4° to 350° C
(39 to 662° F)
Pressure at vents:
~ 250 atmospheres
~ 4,000 PSI
Chemicals:
mM sulfide
High [heavy metals]
pH (acidity):
Similar to vinegar
The hottest water at vents melts plastics, tin & pewter,
chars wood, and dissolves glass and many metals
Hydrothermal Vents
 Vents and their associated microbial communities play a
major –unconstrained- role in marine biogeochemical
cycles
 Sulfur, iron, manganese, copper, nickel, more…
 Influences ocean productivity
 After ~34 years of research, microbial influence on marine
biogeochemistry is largely unconstrained
 Difficult to conduct in situ chemical measurements and
experiments
 Difficult to replicate conditions in the lab
Sulfides host substantial endolithic microbial communities
 Vents host substantial
endolithic microbial
communities (Schrenk et al, 2003)
 From outside to inside:

H2S oxidizing bact., arch.

Sulfate reducing bacteria

Iron/Sulfur reducing archaea

Methanogenic archaea

Most thermotolerant
@122 C
 Numerous undescribed
ribotypes
Tackling a longstanding paradox of vent microbial
ecology and physiology
 Rates of sulfide oxidation in chimneys are
too high; inconsistent with geochemistry,
thermodynamic constraints
 Vent effluents oxidant limited
 One alternative: accessing remote
oxidants
 Metal sulfides (pyrite, wurtzite
chalcopyrite) are highly
(semi)conductive
 Extracellular electron transfer?
How do bugs breathe rocks or access remote oxidants?
Microbial extracellular electron transfer (EET)
Aerobic respiration
Microbial anaerobic
respiration via EET
“food” + O2
your cells
CO2
FeO(OH)·nH2O
(“rust”)
“food”
microbes
CO2
Fe (II)
Microbial fuel cells (MFCs):
A tool for studying microbial EET
 MFCs “mimic” mineral oxidants
 MFCs and potentiostats deployed in situ
or in lab
H2O
O2
e-
“cathode”
 An analogue for the reduction of
mineral oxides
 Microbes shuttle reducing equivalents
to poised anode
 Electrical current
metabolism
“anode”
microbial
H2S, H2, other?
e-
microbes
oxidized S,
H2O
Sulfide “chimneys” span redox gradients
10 cm
 Do vent microbes employ EET
to access remote oxidants?
Oxic
Seawater
(Eh > 0)
Anoxic
fluid
(Eh < 0)
 Chimney surfaces replete with
metal oxides, oxygen
 Sulfides are semi-conductive
 Vary electrical continuity to
oxygen
 Δ electrical current
 Δ microbial diversity, density
 Δ carbon fixation
Anoxic
fluid
H2S
2H+
S0
counter ions
pyrite,
chalcopyrite
 Experiments
2°C
Cross section of sulfide
(Schrenk et al 2002)
porous
chimney
Model of a hydrothermal
vent as a “natural” MFC.
e-
Fe-oxyhydroxides
~150°C
Oxic
seawater
O2
H2O
Artificial “vent sulfide”
to study microbial EET

Conditioning column (yellow)
produces vent-like effluent

Pumped into pyrite chamber

Seawater is pumped into
cathode chamber

Pyrite in electrical continuity
with cathode

Treatments @65 °C




In continuity
No continuity
Δ [H2S] or [H2]
No reductant
Pump
Pump
Vent
Fluid
Seawater
Pyrite
Anode
Graphite
Cathode
inoculation
Artificial “vent” effluent (sulfate-free), 90 °C, pH ≈ 7.2, 3 mM ΣH2S
Current generation did not begin until after inoculation
Post incubation, sampled pyrite and cathode for molecular analyses
Highlights
Pyrite in continuity
with aerobic cathode
 High current
 Substantial biofilm
Pyrite disconnected
from cathode
 No current
 No biofilm
Pyrite with low H2S, H2
 Lower current
 Ample biofilm
Pyrite without H2S, H2
 No current
 No biofilm
Microbes achieve high density on pyrite in continuity
Substantial cell density (~5x106 • cm2), elemental sulfur associated with bugs
NOTE: Pyrite is fully reduced, cannot “accept” more electrons
Electrical continuity yields diverse,
representative community
Anode biofilm diversity
 16s rRNA pyrosequencing of Bacteria
and Archaea
 V6 region; 378MB in total
continuity
 In continuity pyrite hosts diverse
community
 ε and γ proteobacteria dominant
 Archaea recovered, a first for EET
 Without continuity pyrite as low diversity
 δ proteobacteria dominant
 No archaea
NO
continuity
Electrical continuity yields diverse,
representative community
Anode biofilm diversity
IN CONTINUITY =
MICROBIAL COMMUNITIES SIGNIFICANTLY
RESEMBLE ACTIVE VENT COMMUNITY
continuity
NO CONTINUITY =
NO
continuity
(Schrenk et al, 2001; Takai et al, 2003)
MICROBIAL COMMUNITIES SIGNIFICANTLY
RESEMBLE EXTINCT SULFIDE COMMUNITY
(Sylvan et al, 2012)
Probing physiological potential via metagenomics
(454 sequencing, 280MB total)
 Microbes from continuity pyrite show potential for sulfide
oxidation, hydrogen oxidation, carbon fixation
 Microbes from NO continuity pyrite show potential for iron
oxidation, carbon fixation, no archaea
CONTINUITY Pyrite “metagenome”
NO CONTINUITY Pyrite
“metagenome”
Sulfide, thiosulfate oxidation
Sulfur disproportionation
Putative iron oxidizers*
Hydrogen oxidation
Iron scavenging mechanisms
Carbon fixation
(rTCA dominant)
Carbon fixation
(Calvin Benson Bassham)
Quorum sensing and biofilm formation
Type III, IV, VI secretion systems
Archaeal lipid biosynthesis
No archaeal genes recovered
The effect of continuity on carbon fixation
 Repeated experiments with 13C-labeled inorganic carbon
 Incorporation into biomass
CONDITIONS
carbon fixation rate
Pyrite carbon fixation
(nmol • cm2 pyrite day-1)
CONTINUITY
(500µM H2, 3 mM H2S)
354 ± 67
NO CONTINUITY
26 ± 19
CONTINUITY
50µM H2
121 ± 34
CONTINUITY
100µM H2S
195 ± 112
CONTINUITY
No added reductant
BLD
 In continuity, pyrite carbon fixation ~10x higher than no continuity
Vent microbes use EET to donate and accept electrons
(Nielsen and Girguis, in review at Nature)
Iron oxidation
Others?
Carbon fixation
Sulfur oxidation
Methanotrophy
Caminibacter (ε)
Sulfurovum (ε)
SO42- + H+
e- 
Hydrogenimonas (ε)
Sulfurimonas(ε)
Fe (III)
H2S or S°
Thiomicrospira (γ)
Pseudomonas (γ)
Note: abiotic Fe, Hg, and S oxidation as well



abioticFe (II)
Iron
Oxidizers
Biological & biogeochemical consequences of EET
 In oxidant-limited environments, EET
enables microbes to access remote
oxidants (Nielsen et al, 2010)
 EET stimulates primary productivity
 EET reduces local alkalinity

H+ equivalents during microbial EET
 Reshapes our notion of “anaerobic”
metabolism

Evolution of microbial EET may be
driven by oxidant limitation
Current directions: Interrogating global phylogenetic
and functional diversity at hydrothermal vents
 With the DoE-JGI, we are conducting the first comprehensive
phylogenetic survey of vent microbial communities

~150 high and
low temp
samples from
around the
world

Contextual data
for each sample

“open access”
project
 Stay tuned!
Thank you all for your time!
Deep sea hydrothermal vent, 2500 m
Microbial EET mechanisms

Direct e- transfer

Outer membrane
cytochromes
 Electron shuttles

Exogenous or endogenous
shuttles
 Pili and nanowires

Extend many body lengths
S. oneidensis
outer membrane
cytochromes
(K. Nakamura)
P. aeruginosa
pyocyanin
(Hernandez &
Newman, 2001)
S. oneidensis
nanowires
(R. Bencheikh)