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Surface to Lower Biosphere Limit: Long-term Geobiology Reference Transect
Why Biology Needs a DUSEL
Duane P. MoserDuane P. Moser
Desert Research InstituteDesert Research InstituteLas Vegas, NVLas Vegas, NV
Outline:
• Insights and frustrations from prior work
• General concepts to incorporate into design
• Specific ideas for long-term reference transect
Why Long-Term Reference Transect and why DUSEL?
•Almost always sporadic samples of opportunity
• Excavations always done for other purposes
• Very limited capacity for repeat sampling
Learning from persistent challenges from past
SAGMCG1
SAG
MC
G2
Cre
n G
rou
p 1
b
Cre
n G
rou
p 1
cC
ren G
rou
p 2
"Su
bsu
rface" Gro
up
2
Cren Group3
KorarchaeotaYNPFFA
OPA3/4
Thermoprotei
OPA2
Met
hanom
icro
bia
Eukaroyotes
Bacteria
No
rth
am G
rou
p 1WS
A2
Th
ermo
plasm
aH
alo
bac
teri
ap
MG
1 FCG3
Methanobacteria
pMC2
"Sed A
rchaea 1"
FCG1
SAGMEG-1
SAGMEG-2
Archaeoglo
bi
FCG2
Methanococcales
Thermococci
0.10
Marine Group 1
16S rRNA Tree by Thomas Gihring
Long-term Biosustainability in a High-energy, Low-diversity Crustal Biome
Science: Accepted pending revisions
L-H Lin, P-L Wang, D. Rumble, J. Lippmann-Pipke, E. Boice, L. Pratt, B. Sherwood Lollar, E. Brodie, T. Hazen, G. Andersen, T. DeSantis, D.P.
Moser, D. Kershaw, and T.C. Onstott
Brett Tipple, 3.3 kmbls in Mpneng
Service water
Major source of introduced organisms.
Primarily Proteobacteria:Comamonadaceae, Hydrogenophaga, Leptothrix,
Alcaligenes, Nitrosomonas, Rhodobacter, etc.
Drilling fluid
Divergent from service water.
Mostly ProteobacteriaComamonadaceae, Hydrogenophaga, Thiobacillus, Thauera, Pseudomonas, Acenitobacter, Alishewanella, etc.
Borehole fluid, 1 hour:
Most similar to the drilling fluid community.
Introduced community overprints indigenous community.
Primarily Proteobacteria
Borehole fluid, 48 hours:
Still primarily Proteobacteria
Borehole fluids, 30 days:
Drilling fluid and service water communities no longer detected.
Desulfotomaculum and taxa deeply-branched Firmicutes appear.
Borehole fluids, 70 days
Population has stabilized.
7 taxa closely-related to Desulfotomaculum and deeply-branched Firmicutes.
drilling fluid
borehole fluid, 1 hour
borehole fluid, 48 hours
borehole fluid, 30 days
borehole fluid, 70 days
service water
unweighted arithmetic average clustering based on binary, presence/absence distance measures
Bacteroidetes
-Proteo.
-Proteo.
-Proteo.
Nitrospira
OP11
Firmicutes
Synergistes
Percent of clones
1006020
Bacterial 16S rDNA clone distribution
Microbial Community Development in Boreholes
South Africa Subsurface Firmicute Groups (SASFG)
*SASFG-1
SASFG-2
SASFG-3
SASFG-4
SASFG-5
SASFG-6
SASFG-7
SASFG-9
SASFG-8
image courtest of Gordon Southam
Major new bacterial lineages with one exception only found in South African subsurface below 1.5 km depth
Complete genome for SASFG-1 (LBNL). Sulfate reducing, spore former, motile, nitrogen fixer.
Tree by Thomas Gihring
Stable (Indigenous?) Populations
Dec-98
Feb-99
Nov- 2001
Nov-2002
Bacterial T-RFLP data “community 16S rDNA fingerprint (3.2 kmbls Driefontein)”
Isolate DR504
“SASFG-1”
Henderson Reference Transect
•Stable, predictable, platform
•Gold-standard reference site fortesting new technologies
•Deep ecological reserve
•Intact subsurface ecosystem
•“Artificial fracture”
•Track fluid movements (colonization history)
•Repeated sampling
In situ Experiments: Artificial Fracture Zone?
• Stevens and McKinley (H2 production in basalts) controversey… how important are fresh fracture surfaces and how fast do fault surfaces weather… do microbial communities respond to fault slip and other geological disturbances.
•Seismicity: do biofilms lubricate faults?
•Substrates (nutrient stimulation, recoverable mineral coupons)
•Downhole packer
•Multilevel sampler
•U-tube with backfill
•Valve at outlet
Logistics (Hardware)
Operation at ambient pressure?
New systems from industry/DOE (e.g. oil, geothermal)?
• Steel Casings/Valves•Corrosion = failure (stainless?)•Iron source = shifts in population•Hydrogen artifacts
• Plastics/Rubber•PEEK, Delrin? (leaching?, degradation, pressure failure?)•Tubing (nylon, stainless)?
•Titanium?
Logistics (Materials)
• Distance•How far into the rock to escape mining influences?
• Drilling/Coring•Drilling muds (e.g. chemicals, bentonite, introduced bugs)•Rotary drilling with airlift?•Grout
• Legacy oxidation•Minerals oxidized during drilling•Steel cuttings remaining in hole
Logistics (Methods)
• Henderson DUSEL a unique opportunity to finally do subsurface microbiology “right”
• Long-term reference transect would be the gold-standard site for decades and adaptive to new technologies for life detection.
• Different hydrology/lithology at Henderson expands subsurface biomes that will have been explored
Conclusions
Description of experiment: a controlled platform for long-term geobiology laboratory, offering near-continuous coverage of an intact subsurface ecosystem block from shallow-aquifer to near the lower biosphere limit. the tracking of fluid migration in three dimensions and the testing of hypotheses concerning deep microbial colonization history. deep ecological reserve and gold-standard reference site, which could be sampled repeatedly over decades in response to new technologies.
Description of experiment: Roughly ten side-wall boreholes of a minimum 500 m length ea. would be extended horizontally at interval, and into hotter depths by drilling into the mine floor. Holes would be sealed to ambient pressure and outfitted with sampling ports, packers and unreactive multilevel samplers to allow repeated sampling proximal to features and host rock types of interest. Holes in unsaturated zones would be sealed and packered to enable gas sampling and down-hole collection of surface biofilms. Microbial population structure in the boreholes would be assessed using the best available molecular tools, both temporally from time-zero and spatially to quantify the extent and persistence of mining-induced contamination. Facilities would be developed to enable to emplacement and recovery of long-term in situ mineral weathering and substrate addition experiments.
Anaerobic Ecosystems: Life’s Redox Footprint(What would you expect in the very deep subsurface?)
Methanogenesis/Acetogenesis (consume H2)
H20 + CO2
Aerobic Respiration
Nitrate and Mn(IV) Respiration
Fermentations (release H2)
CH20 (Burial)
O2
Sulfate Respiration 1-1.5 nM
0
0.05 nM
H2 concentration
Fe(III) Respiration
7-10 nM
0.2 nM
1) No available respiratory electron acceptors?
A. Witwatersrand quartzite core from 1.95 km depth in fracture zone. Pink = rhodamine tracer. B. 35S auto-radiographic image of core. C. Sulfate reducing bacteria with AgS xtals in pore.
A.
B.
C.
Courtesy of Gordon Southam, Univ. of Western Ontario and Mark Davidson, Princeton University
Endolithic Sulfate Reducers(a shot in the arm for radiolysis)
-Methananobacterium
-Actually an Archaeon (despite the name).
-Makes Methane from CO or CO2 and H2
-Desulfotomaculum
-Well known, sometimes thermophilic sulfate reducer
-Uses acetate, H2, probably CO
But wait a minute…..
Methanogens and sulfate reducers are not supposed to cohabitate!
30 M (radiolytic?) Sulfate
Vast excess (20,000 - 200,000 X) of abiogenic H2
An perfectly-poised, electron acceptor-controlled system?
Table 2. Free energy and steady state free energy flux for possible microbially-mediated reactions. Reactions were modeled frommeasured borehole water constituents.
Redox reaction Process
ΔG( /kJ mol)43 oC
p 9H .0
ΔG( /kJ mol)54 oC
p 9H .0
ΔG( /kJ mol)61 oC
p 7H .8
Power( /kJ c -ell s)
43 oCp 9H .0
Power( /kJ c -ell s)
54 oCp 9H .0
Power( /kJ c -ell s)
61 oCp 7H .8
1) 4C O + 5H2 O => CH4 + 3HCO3
- + 3H+Methanogen esis by
C O disproportionation-260 -257 -233 -8.6x10-14 -9.8x10-14 -1.0x10 -13
2) 4H2 + H+ + HCO3
- => CH4 + 3H2O
Hydrogenotrophi c methanogenesis -70 -63 -68 -6.6 10x -14 -6.9 1x 0-14 -8.6x10-14
3) 3H2 + C O => CH4 + H2O Hydrogenotrophi c methanogenesis -117 -113 -109 -3.9 10x -14 -4.3 10x -14 -4.9 1x 0-14
4) 4H2 + H+ + 2HCO3- =>
Acetat e + 4H2OAcetogenesis -48 -41 -46 -3.9 10x -14 -3.8 10x -14 -5.0 10x -14
5) 4C O + SO42- + 4H2 O =>
4HCO3- + HS- + 3H+ Sulf atereduction -297 -295 -271 -2.0 1x 0-14 -2.3 1x 0-14 -2.5 1x 0-14
6) C O + 2H2 O => HCO3- + H+ +
H2Wate -r shif t reaction -47 -48 -41 -1.6 10x -14 -1.8 10x -14 -1.8 10x -14
7) C O + H2 => 0.5Acetat +e 0.5H+ Acetogenesis -72 -69 -64 -2.4 10x -14 -4.2 10x -14 -1.2 10x -14
8) 4H2 + H+ + SO4
2- => HS- + 4H2O
Sulf atereduction -107 -102 -107 -7.2 10x -15 -7.9 10x -15 -9.7 10x -15
9) Acetat e + SO42- => 2HCO3
- + HS- Sulf atereduction -59 -61 -61 -4.0 10x -15 -4.7 10x -15 -5.5 10x -15
10) Propane+ 2.5SO42-+ 2H+
=> 3HCO3
- + 2.5H2S + H2OSulf atereduction -150 -153 -152 -4.0 10x -15 -4.8 10x -15 -5.5 10x -15
11) Aceta te + H2 O => CH4 + HCO3
- Acetoclastic methanogenesis -22 -23 -23 -3.9 10x -15 -4.6 10x -15 -5.4 10x -15
12 Ethan e + 1.75 SO42-+ 1.5H+
=> 2HCO3- + 1.75H2S +
H2OSulf atereduction -97 -100 -99 -3.7 10x -15 -4.4 10x -15 -5.1 10x -15
13) CH4 + S 4O 2- => H2 O + HCO3- + HS- Anaerobic methan e oxidation -37 -38 -38 -2.5 10x -15 -3.0 10x -15 -3.5 10x -15
CONTRIBUTORS
TC Onstott , Mark Davidson, Bianca Mislowack Princeton U
Jim Fredrickson, Tom Gihring, and Fred Brockman PNNL
Lisa Pratt, Eric Boice Indiana Univ.
Barbara Sherwood Lollar, Julie Ward, Greg Slater U of Toronto
Gordon Southam, Greg Wanger U of Western Ontario
Ken Takai JAMSTEC
Brett Baker UC Berkeley
Tom Kieft New Mexico Tech
Sue Pfiffner, Tommy Phelps U of Tennessee, ORNL
Dave Boone, Adam Bonin, Anna Louise Reysenbach Portland State U
Johanna Lippmann U of Potsdam
Terry Hazen , Eoin Brodie, et al. LBNL
Li-Hung Lin National Taiwan U
Dawie Nel, Walter Seymor, Colin Ralston, etc. etc. Mine professionals
Rob Wilson and staff Turgis Ltd. Consultants
Derek Litterhauer and Esta VanHeerden Univ. of Free State
Chrissie Rey, Faculty, students and staff U of Witwatersrand
2H/18O ratio and other chemistry matches other local waters aged to 3-30 MA
Hydrogen isotope equilibration temp = 60.5 oC e.g. 3 - 5 km source depth
Ca2+/Na+ ratio and other geochem indicates water has not traversed shallower levels (lavas and dolomites)
Thus water most likely aged meteoric, with long flow path, trapped in the Witwatersrand Supergoup (nearest outcrop = 11 km away.
The western Witwatersrand Basin
Ventersdorp lava (Ca2+/Na+ ratio 1.4 )
Witwatersrand quartzite (Ca2+/Na+ ratio 0.12 )
Dolomite (Ca2+/Na+ ratio 2.4 )1 km
2 km
3 km
4 km
5 km
6 km
54 oC temp is higher than geothermal gradient would predict (upwelling)
From Kelly, D.S. et al. 2005, Science, 1428-1434
Lost City (1, 3) Lidy Spring (2)
Columbia R. Basalt (3, 5) D8A (this work)
Marine Continental Continental Continental
Rock peridotite Basalt Basalt quartzite
pH 10 -11 6.9 7.5 - 9.5 9.1
CO2 low 55 mM 1 - 3 mM low
Temp 60 - 75 oC 59 oC 18 - 20 oC 54 - 61oC
CH4 1 - 2 mM 0.1 mM 2 - 209 M 17.5 mM
H2 <1 to 15 mM 0.0013 mM 0 - ca. 80 mM 0.165 mM
sulfate 1 - 4 mM 1.3 mM 0.004 - 1.4 mM 0.03 mM
Dominated Methanogen
SRB (firmicutes)
Methanogen Acetogen? Methanogen
Various SRBs
firmicutes
Methanogen
SRB (firmicutes)
1) Boetius, A. 2005. Science, 307:1420-14222) Chapelle, F. H., . et al. 2002. Nature 415:312-3153) Fry, N. K., J. K. Fredrickson, S. Fishbain, M. Wagner, and D. A. Stahl. 1997. Appl. Environ. Microbiol. 63:1498-1504.4) Kelly, D.S. et al. 2005, Science,307: 1428-14345) Stevens, T. O., and J. P. Mckinley. 1995. Science 270:450-454