Surface to Lower Biosphere Limit: Long-term
Geobiology Reference Transect
Why Biology Needs a DUSEL
Duane P. Moser
Desert Research Institute
Las 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?
Learning from persistent challenges from past
•Almost always sporadic samples of opportunity
• Excavations always done for other purposes
• Very limited capacity for repeat sampling
The Witwatersrand Deep Microbiology Project
(1997-2003)
TC Onstott and many, many others
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
Why Long-Term ?
Hole EB5
Evander Mine
Microbial Community Development in Boreholes
service water
drilling fluid
borehole fluid, 1 hour
borehole fluid, 48 hours
borehole fluid, 30 days
unweighted arithmetic average clustering based on binary,
presence/absence distance measures
borehole fluid, 70 days
Bacterial 16S rDNA clone distribution
Borehole
Borehole
Borehole
Service
Drilling
fluid,
fluids,
fluids,
fluid,
water
fluid
48
130
70
hour:
hours:
days:
days
a-Proteo.
Population
Divergent
Most
Drilling
Major
similar
source
fluid
from
has
toof
and
the
service
stabilized.
service
introduced
drilling
water.
water
fluid
organisms.
communities
community.
Stilllonger
primarily
Proteobacteria
no
detected.
Primarily
Mostly
Introduced
7
taxa closely-related
Proteobacteria
Proteobacteria:
communitytooverprints
Desulfotomaculum
indigenous
Comamonadaceae,
Hydrogenophaga,
Leptothrix,
Comamonadaceae,
community.
Desulfotomaculum
and
deeply-branched
and
Hydrogenophaga,
Firmicutes.
taxa deeply-branched
Alcaligenes,appear.
Nitrosomonas,
Rhodobacter, etc.
Thiobacillus,
Firmicutes
Thauera, Pseudomonas,
Acenitobacter,
Primarily
Proteobacteria
Alishewanella, etc.
b-Proteo.
g-Proteo.
Nitrospira
OP11
Firmicutes
Bacteroidetes
Synergistes
20
60
Percent of clones
100
South Africa Subsurface Firmicute Groups (SASFG)
image courtest of Gordon Southam
SASFG-6
SASFG-5
SASFG-4
SASFG-7
SASFG-3
SASFG-9
SASFG-8
*
SASFG-1
SASFG-2
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
“SASFG-1”
Dec-98
Feb-99
Nov- 2001
Nov-2002
Isolate DR504
Bacterial T-RFLP data “community 16S rDNA fingerprint (3.2 kmbls Driefontein)”
Henderson Reference Transect
•Stable, predictable, platform
•Gold-standard reference site for
testing 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)
Interface Between Oxic and Anoxic World
Logistics (Hardware)
•Downhole packer
•Multilevel sampler
•U-tube with backfill
•Valve at outlet
Operation at ambient pressure?
New systems from industry/DOE (e.g. oil, geothermal)?
Logistics (Materials)
• 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 (Methods)
• 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
Biology DUSEL: Critical or Merely Important?
Conclusions
• 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
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 goldstandard 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 longterm in situ mineral weathering and substrate addition
experiments.
Anaerobic Ecosystems: Life’s Redox Footprint
(What would you expect in the very deep subsurface?)
O2
H20 + CO2
Aerobic Respiration
Nitrate and Mn(IV) Respiration
H2 concentration
CH20 (Burial)
0
0.05 nM
0.2 nM
Fe(III) Respiration
1-1.5 nM
Sulfate Respiration
Fermentations (release H2)
Methanogenesis/Acetogenesis (consume H2)
7-10 nM
1) No available respiratory electron acceptors?
A.
Endolithic Sulfate Reducers
(a shot in the arm for radiolysis)
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.
C.
B.
Courtesy of Gordon Southam,
Univ. of Western Ontario and Mark
Davidson, Princeton University
Driefontein Consolidated Gold Mine
-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
D8A microbial population
But wait a minute…..
Methanogens and sulfate reducers are not supposed to cohabitate!
30 mM (radiolytic?) Sulfate
Vast excess (20,000 - 200,000 X) of abiogenic H2
An perfectly-poised, electron acceptor-controlled system?
Table 2. Free energy and st eady state free energy flux for possi ble microbially-mediated reactions. Reactions were modeled from
measured borehole water constituents.
G
G
G
Power
Power
Power
(kJ/mol) (kJ/mol) (kJ/mol) (kJ/cell-s) (kJ/cell-s) (kJ/cell-s)
o
o
o
o
o
o
43 C
54 C
61 C
43 C
54 C
61 C
Process
pH 9.0
pH 9.0
pH 7.8
Redox reaction
pH 9.0
pH 9.0
pH 7.8
1) 4CO + 5H2O => CH 4 +
Methanogenesis by
-14
-14
-260
-257
-233
-8.6x10
-9.8x10
-1.0x10 -13
3HCO3- + 3H+
CO disproportionation
+
2) 4H2 + H + HCO3 => CH 4 +
-14
-14
-14
Hydrogenotrophic methanogenesis
-70
-63
-68
-6.6x10
-6.9x10
-8.6x10
3H2O
3) 3H2 + CO => CH 4 + H2O
Hydrogenotrophic methanogenesis
-117
-113
-109
-3.9x10-14 -4.3x10-14
-4.9x10-14
+
4) 4H2 + H + 2HCO3- =>
Acetogenesis
-48
-41
-46
-3.9x10-14 -3.8x10-14
-5.0x10-14
Acetate + 4H2O
25) 4CO + SO4 + 4H2O =>
-14
-14
-14
Sulfate reduction
-297
-295
-271
-2.0x10
-2.3x10
-2.5x10
+
4HCO3 + HS + 3H
6) CO + 2H2O => HCO3- + H+ +
-14
-14
-14
Water-shift reaction
-47
-48
-41
-1.6x10
-1.8x10
-1.8x10
H2
7) CO + H2 => 0.5Acetate +
-14
-14
-14
Acetogenesis
-72
-69
-64
-2.4x10
-4.2x10
-1.2x10
0.5H+
+
28) 4H2 + H + SO4 => HS +
-15
-15
-15
Sulfate reduction
-107
-102
-107
-7.2x10
-7.9x10
-9.7x10
4H2O
9) Acetate + SO42- => 2HCO3- +
Sulfate reduction
-59
-61
-61
-4.0x10-15 -4.7x10-15
-5.5x10-15
HS 10) Propane+ 2.5SO42-+ 2H+
=>
Sulfate reduction
-150
-153
-152
-4.0x10-15 -4.8x10-15
-5.5x10-15
3HCO3 + 2.5H2S + H2O
11) Acetate + H2O => CH 4 +
Acetoclastic methanogenesis
-22
-23
-23
-3.9x10-15 -4.6x10-15
-5.4x10-15
HCO312 Ethane + 1.75SO42-+ 1.5H+
Sulfate reduction
-97
-100
-99
-3.7x10-15 -4.4x10-15
-5.1x10-15
=> 2HCO3- + 1.75H2S +
H2O
13) CH 4 + SO42- => H2O +
Anaerobic methane oxidation
-37
-38
-38
-2.5x10-15 -3.0x10-15
-3.5x10-15
HCO3- + HS -
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
The western Witwatersrand Basin
d2H/d18O 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
54 oC temp is higher than geothermal gradient would predict (upwelling)
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.
Dolomite (Ca2+/Na+ ratio 2.4 )
1 km
2 km
Ventersdorp lava (Ca2+/Na+ ratio 1.4 )
3 km
4 km
5 km
6 km
Witwatersrand quartzite (Ca2+/Na+ ratio 0.12 )
Lost City (1, 3)
Lidy Spring (2)
Columbia R.
Basalt (3, 5)
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 mM
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)
D8A (this work)
1) Boetius, A. 2005. Science, 307:1420-1422
2) Chapelle, F. H., . et al. 2002. Nature 415:312-315
3) 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: 1428-1434
5) Stevens, T. O., and J. P. Mckinley. 1995. Science 270:450-454
From Kelly, D.S. et al. 2005, Science, 1428-1434