Oxic-Anoxic Transition Zones:
synergy between geochemistry
and (micro)biology
Brian T. Glazer
Postdoctoral Fellow
University of Hawaii
NASA Astrobiology Institute
glazer@hawaii.edu
Presentation Outline
introductory material
Oxic-anoxic transition zones (OATZ)
Sulfur cycling
Presentation Outline
method development
In situ voltammetry – overview, rationale, & examples
Presentation Outline
2 case studies
Cyanobacterial mats
Oceanic hydrothermal vents
Recap and overall conclusions
Oxic-anoxic transition zones
(OATZ)
Atmospheric O2 rose to 21% ~2 billion years ago
Combined influence of slow diffusion through water
and organic matter ensures OATZ persistence
Photosynthesis and chemosynthesis = redox gradients
Redox gradients = respiration & metabolism
Life is a big, constant, redox equation
Some perspective
• Life history on earth is overwhelmingly microbial
• The earth is ~4.5 billion yrs old,
– microbes arose ~3.5 billion years ago (bya)
– animals-0.7 bya -- humans-0.001 bya
Jan. 1-Earth Forms
The Microbial Age-3.1 Billion Years
Jan
Feb
Late Feb-Microbes
Mar Apr
May Jun
Jul
Aug Sep
Oct
Nov Dec
~Nov. 5th-Animals (oceans)
Dec. 11th-Land Plants
Dec. 27th-Mammals
Dec. 31st- 10:00 PM Humans
11:59:30 PM Written History
Types of metabolism
• Light is used
directly by
phototrophs
• Hydrothermal
energy is utilized
mainly via heatcatalyzed
production of
reduced
inorganics
Nealson and Rye 2004
Oxic-anoxic transition zones
(OATZ)
O2
NO3Mn++
Fe++
SO42-
S2CH4
Nealson and Stahl 1997
NH4+
Redox profiling
General guideline for
OATZ progression
Vertical scale changes across
environments
has been traditionally
oversimplified to SO42and H2S
Sulfur redox cycling
general overview
S0
elemental
sulfur
H2S
hydrogen
sulfide
Sulfate reduction
(dissimilatory)
organic S
Megonigal et al. 2004
SO42sulfate
Sulfur redox cycling
significance
Dissimilatory sulfate reduction:
SO42- + 2CH2O  2HCO3- + HS- + H+
Accounts for half or more of the total organic carbon
mineralization in many environments
Highly reactive HS- is geochemically relevant because of
its involvement in precipitation of metal sulfides and
potential for reoxidation
Sulfur redox cycling
important Fe/S chemistry
H2S oxidation (ph >6):
O2 + Fe2+  Fe3+
Fe3+ + H2S  Fe2+ + S(0) as S8 and Sx2FeS formation and dissociation:
Fe2+ + H2S  FeSaq + 2H+
(FeSaq formation is enhanced with increasing temperature)
Pyrite formation:
FeSaq + H2S  FeS2 + H2
or under milder reducing conditions:
FeS(s) + S2O32-  FeS2 + SO32-
Sulfur redox cycling
important Mn/S chemistry
Manganese catalytic cycling:
Mn2+ + ½ O2 + H2O  MnO2 + 2H+
MnO2 + HS- + 3H+  Mn2+ + 2H2O + S(0) as S8 and Sx2-
Sulfur redox cycling
partially oxidized intermediates
Other pathways for S oxidation:
HS- + 2O2  SO42- + H+
5HS- + 8NO3-  5SO42- + 4N2 + 3OH- + H2O
HS- + 2CO2 + 2H2O  SO42- + 2CH2O + H+
S2O32- + H2O  SO42- + HS- + H+
4S0 + 4H2O  SO42- + 3HS- + 5H+
4SO32- + H+  3SO42- + HS-
Methods & analytical evaluation
electrochemistry
“the science of the interaction of electrical and
chemical phenomena”
…blurring the lines between methods & results…
Voltammetry 101
100 mm gold wire sealed in
PEEK or glass using
marine epoxy, plated with
mercury
O2, Fe2+, Mn2+, H2S,
H2O2, I-, Sx2-, S2O32-,
FeSaq, Fe(III) are all
measurable in one scan,
if present
Au wire – 100mm diameter
Polished epoxy surface
Voltammetry 101
H2O2 + 2H+ + 2e-  H2O
O2 + 2H+ + 2e-  H2O2
Argon purged
Glazer et al. 2004
In situ voltammetry
analytical comparison
250
O2 (mM)
200
150
100
50
electrode
optrode
--- saturation
0
00:00
12:00
24:00
36:00
Elapsed Time (hh:mm)
Glazer et al. 2004
48:00
In situ voltammetry
analytical comparison
275
250
O2 (mM)
225
200
electrode
optrode
Winkler
YSI meter
saturation
175
150
125
00:00
03:00
06:00
09:00
Time (hh:mm)
Glazer et al. 2004
12:00
In situ voltammetry
analytical comparison
230
O2 (mM)
220
210
200
190
electrode
180
00:00
01:00
optrode
02:00
Time (hh:mm)
Glazer et al. 2004
YSI meter
03:00
04:00
In situ voltammetry field application
Coastal bays and sediments
Luther et al. 2004, Taillefert et al. 2002, Rozan et al. 2002,
In situ voltammetry field application
Hydroelectric power generation
Luther et al. 2003
In situ voltammetry application
Laboratory microbial cultures
Sobolev et al. 2001, Roden et al. 2004
In situ voltammetry field
application
Black Sea - world’s largest anoxic basin
photo: Murray, 2003
In situ voltammetry field
application
microbial mats - steep gradients
photo: Glazer, 2002
In situ voltammetry field application
Hydrothermal vents
Riftia
plume
electrode
photo: Glazer, 2003
In situ voltammetry
advantages
1) no need to collect samples
2) a small amount of analyte is used, allowing
multiple measurements
3) microelectrodes can be used to obtain a high
spatial resolution
In situ voltammetry
advantages
4) relatively high data acquisition in a short
period of time
5) electrodes can be deployed in a variety of
water column, sediment, or laboratory
environments
6) simultaneous detection of several analytes
In situ voltammetry
disadvantages
1) labor intensive construction, preparation,
maintenance, and data interpretation
2) expensive
3) relatively high data acquisition in a short
period of time (is actually a two edged sword)
Microbial Mats
steep gradients
2 bya gunflint chert, Michigan
Wisconsin & Australia
Hamelin Bay, Australia
Microbial Mat OATZ
Questions:
Are transient partially-oxidized sulfur
intermediates a measureable component of
the redox transition?
How is the redoxocline affected by the diurnal
cycle?
How are the geochemical gradients reflected by
the microbial consortia?
Microbial Mat OATZ
Technique:
In situ voltammetry should give resolution &
sensitivity required to identify partiallyoxidized sulfide intermediates and other
redox analytes.
DGGE, sequencing analysis, and metabolic
gene-specific PCR will allow for community
characterization on the same vertical scale as
the in situ profiles.
CO2
CO2
O2
H2S
O2
Mat Surface
cyanobacteria
CH2O
CH4
SO4
O2
aerobic
heterotrophs
chemolithotrophic
S-bacteria
phototrophic
S-bacteria
HS-
Sx2-, S8
SO4
FeS
fermenters
Organic acids
& H2
FeS2 precipitation
& burial
sulfate/sulfur
reducers
methanogens
CO2
CH4
Field deployments
Profile A
Time Series
Profile B
Height (m above MLLW)
1.2
1.0
0.8
0.6
0.4
0.2
15:00 18:00 21:00 00:00 03:00 06:00 09:00 12:00 15:00
02 July 2002
Glazer et al., 2004
03 July 2002
Redox speciation (surface)
200
180
160
H2O2
Current (nA)
140
O2
120
100
80
60
40
20
0
-20
-2.0 -1.8 -1.6 -1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0
Volts vs. Ag/AgCl
Glazer et al., 2004
Redox speciation (-0.5 mm)
1000
Current (nA)
800
600
400
200
H2O2
0
-200
-400
-2.0 -1.8 -1.6 -1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0
Volts vs. Ag/AgCl
Glazer et al., 2004
Current (nA)
Redox speciation (-2.0 mm)
160
140
120
H2O2
S0 (S8) O2
100
80
60
40
20
0
-20
-40
-60
AVS
-80
-100
-2.0 -1.8 -1.6 -1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0
Volts vs. Ag/AgCl
Glazer et al., 2004
Redox speciation (-4.0 mm)
Current (nA)
S0(Sx2-)
2-
2-
S (H2S, Sx )
800
700
600
-1
FeS
2000
mVs
500
400
300
S4O62200
100
0
-100
-200
-300
-400
-500
-600
AVS
-700
-2.0 -1.8 -1.6 -1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0
Volts vs. Ag/AgCl
Glazer et al., 2004
Redox speciation (-6.0 mm)
S2- (H2S, Sx2-)
600
500
400
FeS
2000 mVs-1
Current (nA)
300
200
100
0
-100
-200
-300
-400
AVS
-2.0 -1.8 -1.6 -1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0
Volts vs. Ag/AgCl
Glazer et al., 2004
Redox profiles
0
200
400
600
800
0
O2 & H2O2 (mM)
0
100
200
300
FeS current (nA)
FeS curent (nA)
FeS current (nA)
200
400
600
800
0
0
100
200
300
400
600
800
O2 & H2O2 (mM)
O2 & H2O2 (mM)
400
200
400
0
100
200
300
400
2
C.
B.
A.
1
0
Depth (mm)
-1
O2
H2O2
O2
H2O2
-2
0
2-
0
0
2-
0
2-
FeSnA
-6
-7
-8
100 200 300 400 500
Glazer et al., 2004
810
0
100 200 300 400 500
840
Sulfur Species (mM)
2-
S (H2S, (Sx ))
H2S
AVS
-5
0
2-
S (Sx )
2-
S (H2S, Sx )
H2S
AVS
FeS
2-
S (H2S, Sx )
H2S
AVS
FeS
-4
S2O3
2-
S (Sx )
2-
S (Sx )
2-
2-
S8
S4O6
-3
O2
H2O2
0 100 200 300 400 500 600
1000
Spatial
variability
Vertical heterogeneity of up to 1
mm may account for disparity
between geochemistry &
microbial sampling
Glazer et al., 2004
Diurnal (& tidal)
variability
four-electrode array:
surface,
-2.0 mm,
-4.0 mm,
-6.0 mm
Glazer et al., 2004
Surface
Surface oxidation
& dehydration,
20:00
Tidal inundation,
13:00
Glazer et al., 2004
- 2.0 mm
Surface oxidation
& dehydration,
20:00
photoinhibition,
12:00
photosynthesis,
06:50
Glazer et al., 2004
- 4.0 mm
More oxidized,
09:30
More reduced,
22:00
Overnight precipitation of
FeS & FeS2
Glazer et al., 2004
- 6.0 mm
Partially oxidized,
10:00
Reduced, Overnight precipitation of
22:40
FeS & FeS2
Glazer et al., 2004
Polysulfide dynamics (-4.0 mm)
FeS
S0 (Sx2-)
S2- (Sx2-, H2S)
Glazer et al., 2004
Geochemistry recap
Speciation:
polysulfide, thiosulfate, tetrathionate, elemental
S, sulfide, FeS all present in mat
partially oxidized sulfur found in deep portions
of the mat, suggesting sulfur
disproportionation
Spatial & temporal variability:
Heterogeneity in mat layers can result in nonuniform chemical gradients on the order of up
to 1 mm
Surficial atmospheric exposure & photosynthesis
significantly oxidize only the upper 2 mm of
the mat
Microbial community composition
Nucelic acid extraction
DGGE
sequencing
metabolic
gene-specific
PCR
Microbial community composition
young, ephemeral
mat mainly
monoculture
Band D only
present in top
2.2 mm
Glazer et al., 2004
Microbial community composition
Glazer et al., 2004
Microbial community composition
sequence analysis
Band
Most closely related
bacterial sequence
% Sequence
similarity
Length of
sequence
Accession #
-0.2 D
Amphora delicatissima
99.2
132
ACU96445
plastid
-2.2 F
Purple bacterium
(unnamed) str. Thd2
95.8
167
PBTHD216S
-3.2 C
Bacterium K2-15
93.3
135
AY345434
-5.2 G
uncultured a
proteobacterium
99.2
128
Y0944961
Metabolic gene presence
superoxide dismutase:
sodA encodes for Mn-SOD, which acts as the primary
antioxidant defense in many prokaryotes
Rubisco:
cbbL encodes for ribulose 1,5-bisphosphate
carboxylase/oxygenase, which fixes CO2 in the Calvin
cycle
dissimilatory bisulfite reductase:
dsrAB encodes for terminal redox enzyme that catalyzes
the reduction of sulfite to sulfide during anaerobic
respiratory sulfate reduction
Superoxide
dismutase
Rubisco
Glazer et al., 2004
sodA RT-PCR
M
4RT
4N 13RT 13N 23RT 23N 33RT 33N P
N
Glazer et al., 2004
Mat molecular biology recap
Speciation:
Purple sulfur bacteria are present in the mat & likely
are playing a major role in photo-ecology of the
system
Where are the SRBs?
Metabolism:
Concurrent detection of sodA and cbbL down to –3.2
mm, coupled to absence of O2/H2O2 below 2.5
mm suggests light penetration limit & alternate
carbon fixation pathway below –3.2 mm
Hydrothermal Vents
• Discovered 1977
• Heat escapes from
Earth interior
– TH2O = 400oC
– Dissolved chemicals
– Sulfide Precipitation
builds chimneys
QuickTime™ and a
H.263 decompressor
are needed to see this picture.
Video: Alvin Dive 3721: Glazer 2001
Earth’s largest mountain chain
Anatomy of the ridge
Anatomy of a chimney
Anatomy of a chimney
Alvinella pompejana
Photo: Cary, 2001
Alvinella pompejana ectosymbiosis
Cary et al., 1997
Riftia pachyptila
Photo: Lutz, Rutgers, Stephen Low Prod., WHOI
Riftia pachyptila
endosymbiosis
Black Smoker Voltammetry Speciation Data:
0.5 m above vent chimney
free H2S, FeSaq
0.5 m above vent
25 oC; pH = 4.00
25 oC
1.5e-6
1.0e-6
free H2S
FeS
0.5 m
5.0e-7
0.0
360 oC
B
St
-5.0e-7
-1.5
-1.0
-0.5
Volts vs Ag/AgCl
Volts vs Ag / AgCl
Luther et al., 2001
current (A)
Unseen part
of the wand
Current (A)
St = FeSaq + H2S
Pompeii Worm Habitat
Characterization
81°C
20°C
2+
1e-5
FeS
• Major signal due to FeSaq + Fe
• Free H2S/HS- was not detected
• O2 not detected
• Epibionts not chemoautotrophic
2+
No free H2S
D
-1.5
0
St
-1.0
-0.5
Volts vs Ag/AgCl
Volts
vs Ag / AgCl
Luther et al., 2001
Current (A)
Fe
2e-5
current (A)
In Alvinella hole
~80 oC; pH = 5.78
Near Plume of Riftia
8e-8
Riftia field
o
8.5 C; pH = 6.39
Current
current(A)
(A)
6e-8
• H2S/HS- and O2 only
• No FeSaq
• polysulfides can be present
• chemoautotrophs require H2S
Luther et al., 2001
free H2S
4e-8
H 2O 2
O2
2e-8
0
-1.5
-1.0
-0.5
Volts vs Ag/AgCl
Volts vs Ag / AgCl
“Rusty” Riftia
6e-8
"rusty" Riftia 8.6 oC; pH 7.5
Current (A)
current (A)
4e-8
H2O2
2e-8
O2
0
-1.5
• Near ambient conditions
• O2 only dominant signal
• Tubes encrusted with Fe (III)
• NO LIVING TUBEWORMS
Luther et al., 2001
-1.0
-0.5
Volts vs Ag/AgCl
Volts vs Ag / AgCl
The bottom line
Oxic-anoxic transitions
There is a principle difference between gradients of
compounds used for biomass synthesis and those
needed for energy conservation, such as oxygen.
Nutrient limitation leads to a decrease of metabolic
activity, but absence of an energy substrate causes
a shift in the composition of a microbial
community or forces an organism to switch to a
different type of metabolism.
Brune et al. 2000
The after-slides
“I’m glad you asked that…”
In situ voltammetry
reactions & detection limits
Ep (E1/2)
(V)
1a
1b
O2 + 2H+ + 2e-  H2O2
H2O2 + 2H+ + 2e-  H2O
2a
HS- + Hg  HgS + H+ + 2e-
2b
HgS + H+ + 2e-  HS- + Hg
3a
S0 + Hg  HgS
3b
HgS + H+ + 2e- HS- + Hg
-0.30
-1.2
MDL
(mM)
5
5
adsorption onto Hg < 0.60
~ -0.60
< 0.1
adsorption onto Hg < 0.60
~ -0.60
< 0.1
In situ voltammetry
reactions & detection limits
Ep (E1/2)
(V)
4a
Hg + Sx2-  HgSx + 2e-
4b
HgSx + 2e-  Hg + Sx2-
4c
Sx2- + xH+ + (2x-2)e-  xHS-
5
2 RSH  Hg(SR)2 + 2H+ + 2e-
6
2 S2O32- + Hg  Hg(S2O3)22- +
2e-
MDL
(mM)
adsorption onto Hg < 0.60
~ -0.60
< 0.1
~ -0.60
< 0.1
typically more positive
than H2S/HS-0.15
10
In situ voltammetry
reactions & detection limits
Ep (E1/2)
(V)
-0.45
MDL
(mM)
15
7
S4O62- + 2e-  2 S2O32-
8
FeS + 2e- + H+  Fe(Hg) +
HS-
-1.1
molecular
species
9
Fe2+ + Hg + 2e-  Fe(Hg)
-1.43
10
10
Fe3+ + e- Fe2+
-0.2 to -0.9 molecular
species
In situ voltammetry
reactions & detection limits
11 Mn2+ + Hg + 2e-  Mn(Hg)
Ep (E1/2)
(V)
-1.55
MDL
(mM)
5
12
Cu2+ + Hg + 2e-  Cu(Hg)
-0.18
< 0.1
13
Pb2+ + Hg + 2e-  Pb(Hg)
-0.41
< 0.1
14
Cd2+ + Hg + 2e-  Cd(Hg)
-0.58
< 0.1
15
Zn2+ + Hg + 2e-  Zn(Hg)
-1.02
< 0.1
The Nernst Equation
aA + bB = cC + dD
Ecell = Eocell
RT
nF
[C]c [D]d
ln
[A]a [B]b
SO42sulfate
SO32sulfite
S
SO32-
tetrathionate
S
S
SO3
SO32-
thiosulfate
S
elemental
S8
sulfur
0
S
S2Sx
hydrogen
polysulfide
HSsulfide
H2S
sulfur oxidation state
-2
-1
0
1
2
3
4
5
6