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Executive Summary

Introduction. During the first six months of funding
from the NASA Astrobiology Institute (NAI), three major research projects were
initiated by the Indiana-Princeton-Tennessee Astrobiology Initiative (IPTAI) Team: 1. Geomicrobiology and hydrogeochemistry
of intra- and sub-permafrost water intersections in a deep gold mine, Kinross
Luipn Mine, Nunavat Territories, Canada; 2. Partitioning of sulfur isotopes
during pyrite oxidation coupled to radiolytic water cleavage; and 3.
Whole-genome sequencing of an uncultured Desulfotomaculum-like organism
from hydrothermal waters in deep gold mines, Witwatersrand basin, South
Africa. Each of these projects requires a high degree of collaboration among
IPTAI laboratories and involves two to four principal investigators, one to
three post-doctoral associates, and two to five graduate and undergraduate
students.

The central focus of the IPTAI team is investigation of
psychrophilic microbial communities in a deep gold mine located near the Arctic
Circle in northern Canada.

Figure 1. Headframe and living quarters (red buildings) at Kinross Lupin Gold Mine, Nunavat Territories, Canada. View of the mine from the winter haul road in May 2004.

This project involves highly instrumented field and laboratory activities (Figure 2) that build on expertise developed during 5 years of work on thermophilic microbial communities in the deep and ultra-deep gold mines of South Africa. Intra-permafrost/sub-permafrost brines from Arctic and Antarctic regions are important terrestrial analogues for Martian groundwater. Delineating biogenic signatures in analogue environments is crucial for designing life-detection probes for deployment on future Mars exploration missions.

Figure 2. Tullis Onstott (IPTAI-Princeton) and Monique Hobbs (Ontario Power Generation) working to install a noble gas diffusion sampler at the 890 meter level, Lupin Mine.

1.
Geomicrobiology and hydrogeochemistry of intra- and sub-permafrost water
intersections in a deep gold mine, Nunavat Territories, Canada.
PIs Onstott, Pratt, Sherwood-Lollar, Clifford, and
Pfiffner. Distinct microbial communities are anticipated in brines
associated with continuous permafrost but uncontaminated samples of permafrost
brines are rarely available for scientific study. The main shaft, ore workings,
and exploration drifts at Lupin mine allow controlled study of both intra- and
sub-permafrost waters. Water intersections at Lupin are located in fracture
zones hosted by low-permeability Archean metagraywacke, phyllite, and banded
iron formation. An initial field trip to assess the scientific potential at
Lupin was conducted in May 2004 when Pratt and Onstott from IPTAI and Corien
Bakermans from Michigan State University (MSU) joined an international team of
permafrost investigators for collection of water and rock samples (Figures 3
and 4). A second sampling trip is planned for fall 2004. Samples from the May
2004 field trip will be given to the Marine Biological Laboratory (MBL) for
assessment of eukaryotic activity. It is particularly exciting to link IPTAI’s
deep-subsurface expertise with MSU’s resources as a center for research on
psychrophilic microorganisms from Siberian permafrost and Antarctic lake ice
and MBL’s resources as a center for research on extremophile eukaryotes.

Figure 3. Tullis Onstott (IPTAI-Princeton) innoculating iron-reducing media with subsurface brine at the 1130 meter level, Lupin Mine.

Figure 4. Corien Bakermans (MSU lead team) injecting subsurface brine into a sterile serum vial filled with nitrogen, Lupin Mine.

With the assistance of Timo Ruskeeniemi (Geological Survey
of Finland) and Monique Hobbs (Ontario Power Generation), brines of widely
varying salinity were collected from 11 subsurface sites in May 2004. Brines
below the permafrost were collected from six drill holes outfitted with valves
and pressure gauges and located at the 1130- and 880-m levels. Dripping water
from open fractures in the roof was collected at the 1130- and 250-m levels.
Dripping water at the 250-m level is within the current permafrost. Water
recirculated within the mine for drilling activities (service water) was
sampled from an open drain at the 1130-m level. The following types of
aliquots were collected:

a. Filters for DNA analyses.

b. Filters for enrichment of
cultivable, anaerobic and aerobic psychrophiles.

c. Anaerobic media (sulfate
reducers, fermenters, Fe (III) reducers and methanogens) inoculated with
borehole water.

d. Gas samples for isotopic
analyses and measurement of dissolved H₂ and CO.

e. Water sample for isotopic
analyses, including N isotopes of NH₄⁺ (Waterloo)

f. Noble gas samples (Ottawa).

g. Dissolved sulfate/sulfide
samples for isotopic analyses.

h. Samples for FISH (Fluorescent
In Situ Hybridization) and flow cytometer (cell density)

i. Multiple samples for analyses
of cations, anions, and short-chain fatty acids.

Field parameters, pH, Eh, dissolved O₂,
temperature and conductivity were measured on site. Conductivity ranged from
4.9 to 60.4 mS/cm. Temperatures varied from 13.4 to 1.5^oC. The pH
ranged from 7 to 9. Visible ZnS precipitates were observed in the sulfur
isotope syringes after one day of storage at room temperature indicating the
presence of significant sulfide concentrations. Although Eh values as low as
-143 mV were measured, Eh values were higher than expected for a
sulfate/sulfide redox couple at the observed pH and temperature. One possible
explanation is that mixing of water with different reduction potentials from
separate fractures is occurring within boreholes. Televiewer logs indicate the
presence of multiple fractures, and fracture water chemistry seems highly
variable based upon observations of seeps.

The integrity of borehole installations at Lupin is a tribute
to the technical ability of the Finish/Canadian collaborators on the Permafrost
at Lupin Project. We know of no equivalent borehole array available for
scientific study at a deep mine. Of particular interest for sampling deep
subsurface microbes, there are high in situ water pressures (300 to 700
psi), sulfide is present, dissolved O₂ is absent, and the boreholes
have been isolated for time periods up to 14 months. These are important
positive indicators that the borehole microbial communities represent
indigenous organisms in the rock formation. Over the next several months, the
sulfur isotopic and DNA analyses combined with the results of microbial
enrichments will allow assessment of the extent of potential mining
contamination of the environment and to target specific boreholes for more
detailed sampling via a borehole packer system.

2. Partitioning of sulfur isotopes during pyrite
oxidation coupled to radiolytic water cleavage. PI’s Ripley, Sherwood-Lollar,
Pratt, and Onstott. A series of experiments are designed to investigate
sulfur isotopic effects that may accompany the oxidation of sulfide minerals
initiated by reaction with products formed during the radiolysis of water
molecules. Radiolysis produces elevated H₂ concentrations in waters,
as well as strong oxidants such as H₂O₂ and O₂.
Both H₂O₂ and O₂ may react with sulfide
minerals as S^o, S₂O₃^2-, or SO₄^2-.
Thiosulfate (S₂O₃^2-) can undergo
disproportionation to produce both oxidized (SO₄^2-) and a
reduced (S^2-) sulfur species. Because of the isotopic fractionation
between reduced and oxidized species, large )(sulfate-sulfide)
values may be a result of radiolytic oxidation processes. Sulfate-reducing
bacteria could utilize the oxidized sulfur species with the ultimate production
of sulfide minerals. However, the generation of sulfide minerals from the
reduced species liberated from thiosulfate could be characterized by *³⁴S values that are
similar to those produced in sulfide minerals where the reduced sulfur is of
biologic origin. We must understand the potential sulfur isotopic effects that
are associated with each of these processes.

To this end we have been involved in designing a methodology
to recover both reduced and oxidized sulfur species from thiosulfate. To date,
32 experiments to evaluate the sulfur isotopic mass balance in the
disproportionation of Na₂S₂O₃ have been
completed. A thiosulfate solution is formed by mixing powdered sodium
thiosulfate (Na₂S₂O₃) with deoxygeneated,
nanopure water under a N₂ atmosphere. The thiosulfate solution has
been reacted with silver nitrate (AgNO₃) for periods varying from 1 to 2 hours; this step leads to the recovery of reduced sulfur as Ag₂S. After
the removal of Ag₂S (Figure 5) by filtration, the residual solution
is heated to 80^EC and
Ba(NO₃)₂ is added to produce BaSO₄. Initial
experiments utilized BaCl, but excessive amounts of AgCl were formed and
prevented a quantitative recovery of BaSO₄. Isotopic measurements
are in progress to determine the most efficient protocol to accurately
characterize the distribution and isotopic fractionation of sulfur species.
This work constitutes a portion of the Ph.D. research of Irene Arango at Indiana University.

Figure 5. Nitrogen-purged extraction apparatus for quantitative recovery of dissolved and volatile sulfur species, Biogeochemical Laboratories, Indiana University.

In addition to the experiments on thiosulfate and its
disproportionation products we will load our first closed-system pyrite
oxidation experiments in July. Hydrogen peroxide will be syringed through a
septum into a Pyrex tube containing finely ground pyrite. The tube will be
sealed and the experimental charge heated for a 1- to 2-week period. Various
temperatures will be utilized to evaluate the kinetics of the pyrite oxidation
reaction.

During the past six months, Indiana University advertised
for our post-doctoral position. Several excellent candidates responded, and
our evaluation and interview process was necessarily thorough. The position
was offered to Ms. Lili Lefticariu who has recently completed her Ph.D.
dissertation involving stable isotopic geochemistry at Northern Illinois University.
Lili will be in Bloomington in early July to discuss the project and will begin
her work in mid-August. Over the coming year, experiments running at Indiana
will be coordinated with similar experiments running at Toronto in order to
study carbon isotope effects on oxidized and reduced carbon species.

3. Whole-genome sequencing
of an uncultured Desulfotomaculum-like organism (DLO) from hydrothermal
waters in deep gold mines, Witwatersrand basin, South Africa. PI’s Brockman, Hazen, and Onstott. A deep-branching clade
of nearly identical DLO sequences (>99% homology) has been identified in 6
boreholes in 4 mining complexes separated by as much as several hundred km.
The DLO has been the dominant bacterium (>90-100% of clones) present in
clone libraries from very high quality sample sets in two different mines, and
their dominance was retained throughout a several month time series in one
case, and a 648-m vertical depth profile in the other case. Sulfate reduction
appears to be the dominant terminal electron accepting process in these sample
sets based on sulfate and sulfide levels, sulfur isotope geochemistry, and
Gibbs free energy calculations. While prediction of physiology from phylogeny
is not straight-forward in this case, the environmental chemistry, the DLO’s
strong dominance in the community, and the DLO’s affiliation with cultivated
Desulfotomaculum spp. that are sulfate-reducers all speak to the likelihood
that it is a sulfate-reducer. The closest cultured relative is Desulfotomaculum
kuznetsovii (90% similarity) and the closest sequence in Genbank is an
environmental clone recently recovered from oceanic crustal fluid (95%
similarity). Multiple attempts by different laboratories to culture the DLO
have been unsuccessful.

To gain an understanding of the capabilities and degree of
genetic novelty of the DLO, we have filtered large quantities of fissure water
from a sample dominated by the DLO in order to provide adequate biomass for
community DNA sequencing. The Department of Energy Production Genomics
Facility will conduct the sequencing in collaboration with Pacific Northwest
National Lab and Lawrence Berkeley National Lab. A total of 35,000 liters
containing 4×10^4 cells per ml were filtered through a large area filter
cartridge. This filter should yield 280 micrograms of DNA assuming a genome
size of 2 Mbp/cell and 10% extraction efficiency. A minimum of 10 micrograms
is needed for shotgun sequencing

DNA was extracted from 15% of the filter and quantified by
spectrophotometry and gel electrophoresis. DNA yield was poor with only 400
nanograms of DNA recovered from 15% of the filter, indicating an extraction
efficiency of only 0.1%. In addition, only 25% of this DNA was of the size (10
kb or larger) needed for shotgun sequencing. To preserve as much filter as possible,
two other extraction protocols were used (as a screen) on smaller aliquots of
the filter to determine if they would produce detectable DNA. No DNA was
detected with these methods. These results indicate that DNA extraction
efficiency is much poorer than expected.

In an effort to optimize the extraction efficiency, 1 x
10^12 Arthrobacter cells (a difficult to lyse Gram positive bacteria
used as a model for the DLO) were filtered through an identical filter
cartridge. A different extraction protocol (using lysozymeàsodium dodecyl sulfate/Proteinase Kà guanidine isothiocyanate, with a liquid
nitrogen and mortar and pestle grinding pretreatment) was used and compared
against the previous extraction method. DNA extraction efficiencies were 11%
with the new method and 0.5% with the original method. In addition, all DNA
was greater than 10 kb in size. The new protocol will be tested on a small
fraction of the filter containing the DLO, and once its performance has been
confirmed to be adequate, one half of the remaining filter containing the DLO
will be extracted.

Education and Public Outreach (EPO). Outreach for
the IPTAI Team is centered at the University of Tennessee and is under the
direction of Susan Pffifner. Funds from NAI were used to match funds from the
National Science Foundation to support a seven-week Research Experience for
Undergraduates (REU) held in South Africa and targeted toward minority
students. The purpose of the South African REI is to engage students in
geomicrobiological research and to encourage students to think about scientific
careers. American students work side by side with South African students under
the joint supervision of U.S. and South African faculty. One Taiwanese, ten
American, and six South African students participated in the summer 2004
program. In addition to REU activities, eight lectures and seven media
interviews were handled by Pratt, Onstott, and Pfiffner during the first six
months of NAI funding for IPTAI . Additional information on EPO activities is
available on the IPTAI website at ( http://www.indiana.edu/~deeplife (.