Transcript Document

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13C
and 14C Studies of Microbial Carbon
Cycling in the Deep Subsurface
Kevin Mandernack
Dept. of Chemistry and Geochemistry
Colorado School of Mines
Golden CO 80401
[email protected]
N
Importance of Life in the Subsurface
1. Groundwater Resource1
•
30% of freshwater is groundwater (0.3% lakes and rivers)
•
Deep pristine groundwater is threatened by percolating
contaminants
2. Radioactive Waste Disposal2
•
131 temporary sites / 39 states
•
105,000 metric tons spent nuclear reactor fuel in U.S. by 2035
3. Origins of Life / Extraterrestrial Life
•
Could life have originated underground?3,4
•
Could there be life in the deep subsurface of Mars?3,5
1World
Meteorological Organization (http://www.wmo.ch)
2Department
3Stevens
of Energy (http://www.ocrwm.doe.gov)
(1997) FEMS Microbiology Reviews
4Pedersen
(1997) FEMS Microbiology Reviews
5McKay et al. (Science) 1996
Research Overview
Statement:
•
Microbes control subsurface aqueous geochemistry
Objective:
•
Characterize microbial communities & carbon cycling
in the subsurface
Approach:
•
Structural analysis of bacterial cell membrane
phospholipid-derived fatty acids (PLFAs)
•
Measure d13C and 14C values of bacterial PLFAs
•
Measure the d13C and 14C values of possible
groundwater carbon sources (DOC, DIC, POM, CH4)
Bacterial Metabolisms:
Heterotrophy
Autotrophy
Methanotrophy
Subsurface Bacterial Metabolisms
Electron
Donors
Electron
Acceptors
Products
Metabolism
CH2O
CH2O
Fatty acids, H2
Fermentation
CH2O
O2
CO2 + H2O
Aerobic
Respiration
CH2O
Fatty Acids
Fe3+
CO2 + Fe2+
Anaerobic
Respiration
CH2O
Fatty Acids
SO42-
CO2 + H2S
Anaerobic
Respiration
Acetate
Acetate
CO2 + CH4
Methanogenesis
H2
CO2
CH4
Methanogenesis
H2
CO2
Acetate
Acetogenesis
Summary of Terminal Electron Accepting Processes
Aerobic
Denitrification
Respiration
Organic
Matter
Oxidation
C2H4O2
CO2
C2H4O2
CO2
Mn(IV)
Reduction
C2H4O2
CO2
Fe(III)
Reduction
C2H4O2
CO2
Sulfate
Reduction
C2H4O2
Methane
Production
CO2
CO2
C2H4O2
2O2
2H2O
1.6NO3-
1.8N2
4MnO2 4MnCO3 8FeOOH 8FeCO3
SO42-
CH4
S2-
Free Energy
(kJ / mol e-)
-106
-98
-77
-22
-6
-4
Predominant
Redox
Species
SO42Fe(III)
Mn(IV)
NO3-, N2
SO42Fe(III)
Mn(IV)
NO3-, N2
SO42Fe(III)
Mn(IV), Mn(II)
NH4+, N2
SO42Fe(III), Fe(II)
Mn(II)
NH4+, N2
SO42-, S2Fe(II)
Mn(II)
NH4+, N2
S2Fe(II)
Mn(II)
NH4+, N2
(<0.1 nM)
(<0.1 nM)
10
Characteristic
Hydrogen
Concentration
(nM) 5
0
Not
Applicable
Lovley and Chapelle (1995) Reviews of Geophysics
Typical Distribution of Terminal Electron
Accepting Processes in Deep Aquifers
Lovley and Chapelle (1995) Reviews of Geophysics
Two Hypotheses for Abiotic H2 Production in
the Subsurface
Radiolysis of Water by Uranium Decay1
235U
231Th
a2+
H2O
H2 + ½O2
Water-Basalt Interaction2
(X/2)H2O + (FeO)X(SiO2)y
basalt
(mafic)
1Pedersen
2Stevens
(1997) FEMS Microbiology Reviews
& McKinley (1995) Science
(X/2)H2 + X(FeO3/2) + Y(SiO2)
Photosynthesis-Independent Subsurface
Ecosystem
H2 Oxidizers
Organic
Polymers
Desulfobacterium(H2 + CO2)
Desulfovibrio (H2 + acetate)
Shewanella (H2 + org. C source)
Methanotrophs
CH4
acetate
acetate
autotrophic
acetogenic bacteria
acetoclastic
methanogens
H2
CO2
“geogas”
Pedersen (1997) FEMS Microbiology Reviews
CH4
Fermenters
Propionibacterium
Clostridium
autotrophic
methanogens
Identification of Bacteria by
Phospholipid Fatty Acid
(PLFA) Analyses
Archaea
O
O
(phosphoether lipids)
O
N
P
O
+
O
-
O
Bacteria
O
O
O
O
O
(phosphoester lipids)
P
cell
membrane
+
O
N
O-
FAME
Structure
16:0
16:1w7
Likely Source
O
CH3
Ubiquitous in bacteria, plants, &
animals
O
CH3
Common in bacteria
O
O
O
18:1w8
O
CH3
Type II Methanotroph Biomarker
CH3
Sulfate Reducers (Desulfovibrio)
O
i15:0
O
O
cy17:0
Taylor & Parkes (1983) Journal of General Microbiology
Kohring et al. (1994) FEMS Microbiology Letters
Pinkart et al. (2002) In: Manual of Environmental Microbiology
CH3
O
Desulfobacter, Methanotrophs,
Stressed Bacteria
Bowman et al. (1991) FEMS Microbiology Ecology
Guckert et al. (1991) Journal of General Microbiology
Schematic of Site Geology & Sampling
~2 km SE
Tono Mine Shaft
Seto Group
Upper
Lower
Lignite
Seams
Mine
Gallery
Toki Lignite- Bearing Form.
Akeyo
Form.
Odiwara
Form.
Uranium
Ore
Bodies
MSB-2
3
4
Iwatsuki et al. (2001)
Applied Geochemistry
1
5
2 KNA-6
Artesian Flow
Toki Granite
Tono Uranium Mine
Toki, JAPAN
Tsukiyoshi
Fault
Iwatsuki & Yohsida (1999)
Geochemical Journal
Sasao et al. (2006) Geochemistry:
Exploration, Environment, Analysis
Experimental Procedures
3.
2.
Total Lipids
1.
Cells on Filters
Phosphoplipid
Isolation Via SPE
5.
a) GC-MS
Structural Information
b) GC-Isotope Ratio MS
c) Accelerator MS
14C
d13C
Values
Mass Spectrometer Analyses
Phospholipids
Filtration (0.2 mm)
of Groundwater or
Bacterial Culture
Bligh-Dyer Total
Lipid Extraction
4.
Fatty Acid Methyl
Esters (FAMEs)
MeOH +
KOH + Heat
Mild Alkaline
Methanolysis
Type II methanotroph
MSB-2 Granite
KNA-6 Sedimentary
PLFA Structure
18:2 (n6)
18:1w7c
18:1w8c
18:1w9c
18:0
cy17:0
16:1w5c
16:1w7t
16:1w7c
16:0
i16:0
15:0
ai15:0
i15:0
KNA-6 Granite
14:0
60
50
40
30
20
10
0
60
50
40
30
20
10
0
60
50
40
30
20
10
0
i14:0
% of Total PLFA
PLFA Profiles of
Tono Area Groundwaters
PLFA Profiles of
Henderson Mine Groundwaters
trace
Sulfate reducers?
trace
D-1
PLFA Structure
18:1w11
18:1w9
18:1w7
18:0
cy17:0
ai17:0
i17:0
10me16:0
16:1w7
16:0
br16:0
15:0
ai15:0
i15:0
D-3 3/9/06
Pre-packer
me15:0
50
40
30
20
10
0
50
40
30
20
10
0
D-4 (higher SO42-)
14:0
% of Total PLFA
50
40
30
20
10
0
Comparison of PLFA-Based and Direct Cell Counts
MSB-2 Granite
KNA-6 Sedimentary
KNA-6 Granite
PLFA-Based
Total Cells
D-4 3/23/06
Direct Count
Total Cells
D-3 3/9/06
PLFA-Based
Methanotrophic
Cells
D-1 3/23/06
0
1
2
3
Log [cells per mL]
4
5
6
Determination of Bacterial Diet
by d13C and 14C of PLFAs
Stable Carbon Isotope Fractionation
Many biologically-mediated reactions prefer 12C
to 13C due to slightly smaller bond strengths
d13CCO2 = -9 ‰
12CO 12
12CO
2 CO2
132CO
13CO
2
2
12CO
12
12
12CO CO CO
212
2
2
2
CO2
13CO
12CO
2
2 12CO
132CO
2
CO2 Fixation
d13Cbiomass = -27 ‰
12CH
2O 12
O
CH2O
2
12CH O
12CH O
12CH12
12CH O
2
O
2
CH
O
2
2
2
12CH O
12CH O12CH
O O2
13
2CH
2
2
13CH O
12
2 CH O
12CH
2
D13Cbiomass-CO2 = d13Cbiomass - d13CCO2 = -18 ‰
Stable Carbon Isotope Signatures of
Carbon Assimilation Pathways
Heterotrophy
Organic
Compounds
(CH2O)
D13C = ~0
CO2
+
Energy
Biomass Carbon
Autotrophy
CO2 + Energy
Calvin
Benson
Others
D13C
=
D13C
=
-10 to -22
0 to -36
Biomass Carbon
Methanotrophy
CH4
Type I, II, & X
Energy + CO2
Type II & X
RuMP
Serine
D13C
=
D13C
=
-14 to -29
-5 to -14
Biomass Carbon
PreuB et al. (1989) Zeitschrift für Naturforschung
Summons et al. (1994) Geochimica et Cosmochimica Acta
Jahnke et al. (1999) Geochimica et Cosmochimica Acta
Hayes (2001) in: Stable Isotope Geochemistry, Reviews in Mineralogy and Geochemistry
d13C and 14C – Two Indicators of Carbon Source
Atmospheric CO2
14C
(pMC)
100
Modern Organic Matter
Modern Carbonates
80
60
40
20
Ancient Organic Matter
Ancient carbonates
0.1
-20
-30
-10
0
atm
CO21
C3 plants2
Algae2
marine
carbonates1
C4 plants2
-110
Methane4
freshwater carbonates1
d 13C (‰)
1Hoefs
(1997) Stable Isotope Geochemistry
3Feux
2Faure
(1986) Principles of Isotope Geology
4Whiticar
(1977) Journal of Geochemical Exploration
et al. (1986) Geochimica et Cosmochimica Acta
+10
Isotopic Values of Carbon Sources in
KNA-6 Groundwaters
Groundwater
Transport
Carbon
Source
d13C
(‰)
DIC
-18
15
CH4
-95
N.M.
Fulvic Acids
-27
31
POM (lignite)
-25
0
14C
(pMC)
and d13C Values of PLFAs From
Tono Area Groundwaters
PM14C = 57
PLFA
MSB-2 Borehole
79-130 m
16:0
16:1 suite
cy17:0
MSB-2 Borehole
132-154 m
PM14C = 59
18:1 suite
PM14Ctotal PLFAs = 33
Tono Mine
MSB-2 Borehole
171-175 m
Tono Mine
-60
-50
-40
-30
-20
d13C (‰ PDB)
-10
SEDIMENTARY
Drill Fluid
0
GRANITE
14C
Conclusions
1.
PLFA analysis indicates diverse bacterial communities in
granite-hosted ground waters at Tono & Henderson Mines.
Results at Henderson suggest the presence of sulfate
reducing Bacteria.
2.
Estimates of cell numbers at Henderson are ~1x104/ml.
3.
d13C values of bacterial PLFAs are generally lower for the
granite rocks relative to sedimentary rocks at Tono Mine,
suggesting that autotrophic metabolism, including
methanotrophy, is more prevalent here. Henderson???
Acknowledgements
•
•
•
•
•
•
Chris Mills & Raleigh Schmidt – CSM
Teruki Iwatsuki, Ph.D. – JNC Tono Geoscience Center,
Japan
Yuki Murakami, Ph.D. – JNC Tono Geoscience Center,
Japan
Bob Dias, Ph.D. – Dept. of Chemistry, Old Dominion
University, Norfolk, VA
Greg Slater, Ph.D. – School of Geography & Geology,
McMaster University, Hamilton, ON
Chris Reddy, Ph.D. – Woods Hole Oceanographic
Institution, Woods Hole, MA
Funding
•
Japan Nuclear Cycle
•
Earth Sciences program of NSF (EAR-9985234)
Remaining
Questions
1. Do aerobic methanotrophs and
methanogens coexist?
2. Where do aerobic methanotrophs get
O2 in a reduced environment?
?????????
Conclusions
1.
d13C values of bacterial PLFAs are generally lower for the
granite rocks relative to sedimentary rocks, suggesting
that autotrophic metabolism, including methanotrophy, is
more prevalent here.
2.
Detection of the diagnostic 18:1w8 PLFA and molecular
evidence for a monooxygenase enzyme indicate that
type II methanotrophs are present in Tono mine
groundwaters.
3.
d13C value of CH4 suggests autotrophic methanogenesis
in KNA-6 groundwaters
4.
Initial 14C analyses of bulk bacterial PLFAs from Tono
Mine water indicate bacteria indeed utilize relatively old
Additional d13C and 14C
Values of Subsurface
Carbon
Sedimentary
d13C
(‰)
14C
(pMC)
DOC Humic
-42
26
DOC Fulvic
-27
31
Average PLFAs
-42
33
DIC -17.4 15 to 31
POM
-25
0
(assumed)
Granite
d13C (‰)
-50
14C
(pMC)
22
-51
-18.2
14 to 29
-25
0
(assumed)
Examples of Archaebacterial & Bacterial Lipids
Sulfate-Reducers
Archaebacteria Lipids
Desulfovibrio sp.
crocetane
(2,6,11,15-tetramethylhexadecane)
HOOC
iso-C15:0 fatty acid
HOOC
PMI
(2,6,10,15,19-pentamethylicosane)
anteiso-C15:0 fatty acid
HOOC
iso-C17:1w7 fatty acid
Methanotrophs
Desulfobacter sp.
H3COOC
HOOC
C16:1w8 (type I)
10-methyl C16:0
H3COOC
HOOC
C18:1w8 (type II)
cyclic C17:0
Makula, 1978; Coleman et al., 1993; Vainshtein et al., 1992; Nicholas et al., 1986; Chappe et al., 1982
Isolation of Specific Compounds for 14C Analysis
• Separate individual
compounds on capillary
GC column
(preparative capillary gas chromatography)
• Collect selected compounds
in cryogenic traps
connected to column by a
computer controlled zerodead-volume multiport
valve
• Repeat injection up to 100
times to collect sufficient
sample for 14C analysis (
100 mg C)
• Autoinjector required for
retention time consistency
Eglinton and Aluwihare, Analytical Chemistry, 1996.
D14C of PLFAs Indicates
Kerogen as Carbon Source
PLFA Values of Kerogen
Enrichment Culture
PLFA
Mass
(nmol/g)
D14C
(‰)
carbon
d13C
(‰)
Fancient
16:0
3.91
-711.3 ± 14.4
0.744
-25.56
18:0
1.78
-773.9 ± 13.8
0.802
-26.19
18:1 + 18:2
5.08 (18:1)
1.16 (18:2)
-882.2 ± 6.0
0.901
-26.50
cy17:0 + cy19:0
1.65 (cy17:0)
2.87 (cy19:0)
-921.6 ± 7.8
0.937
-26.94
Total PLFA Mass
16.45
Kerogen
-990.1 ± 0.8
-29.5
Atmospheric CO2
~ + 100
~ -7
Petsch et al., 2001
13C/12C
= 1.11 %
PeeDee Belemnite (PDB):
atom %
13C
=
13C
13C/12C
= 0.0112372
/ (12C + 13C)
d13C = (Rsam - Rstd) x 1000
Rstd
13C
12C
13C
= 1,001
= 100,000
Rsam = 0.01001
d = +1 ‰
12C
= 1,000
= 100,000
Rstd = 0.01000
Phospholipid Fatty Acids
(PLFAs) of the Cell
Membrane
O
O
O
O
O
+
O
P
N
O
Oaqueous cell exterior
Cell Death
(phospholipase)
hydrophobic membrane
interior
O
O
O
O
aqueous cell interior
J.D. Hendrix http://science.kennesaw.edu
White & Ringleberg, 1997
OH
Dating Subsurface
Carbon Sources With 14C
Percent Modern Carbon (pmc)
Atmospheric 14C
~ 1ppt of total C
100
80
1/2 life = 5730
years
60
40
20
0
0
10
20
30
40
Age (Kyears Before Present)
50
Faure, 1986
www.NOSAMS.WHOI.edu