Pacific Northwest National Lab

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Pacific Northwest National Lab Jim Fredrickson
and Fred Brockman Duane Moser and Tom
Gihring Ken Takai
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• Major Resource Master Phylogenetic Tree (ARB)
• Tom Gihring (Keeper)
• About 3000 16S sequences
• 1277 SA clone types
• Contributed by many
• Room for more

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Microbial and geochemical variables over a 648 m
vertical transect from an upflowing borehole
intersecting a geological discontinuity at 3.4 km
below land surface. "The Dream Borehole" Moser,
D.P., T.M. Gihring, T.C. Onstott, J.K.
Fredrickson, F.J. Brockman, D.L. Balkwill, M.E.
Dollhopf, S. Pfiffner, L.M. Pratt, B. Sherwood
Lollar, G. Southam, A.T. Welty, L.H. Lin, S.
Mccuddy, T. Kieft. Applied and Environmental
Microbiology, in preparation.
Images Gordon Southam
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Bacterial (upper) and Archaeal (lower) T-RFLP for
Dream Borehole
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Tree (Finished) for Dream Borehole 16S rRNA
DR9-38-H3 clone group 1
South African gold mine clone W6-38-SC1-106
South African gold mine clone W6-38-SC1-119
Desulfotomaculum kuznetsovii
Desulfotomaculum australicum
Desulfotomaculum geothermicum
Desulfotomaculum alkaliphilum
Desulfotomaculum ruminis
Peptococcus niger
Low GC Gram Positives
Clostridium thermolacticum
DR9-38-H3 clone group 2
PCE-dechlorinating enrichment clone ST12
Desulfosporosinus orientis
DR9-38-H3 clone group 3
South African gold mine clone E5-46-SC1-141
Thermacetogenium phaeum
Moorella glycerini
Thermoanaerobacter thermocopriae
Syntrophomonas wolfei
Bacillus infernus
Lactobacillus fermentum
Mycoplasma capricolum
Pseudomonas spinosa
South African gold mine clone E5-46-HS3P-325
Comamonas testosteroni
Commamonadacaea
DR9-38-H3 clone group 4
"Leptothrix discophora"
"Sphearotilus natans"
DR9-38-H3 clone group 5
South African gold mine clone E5-46-BH1-12/7/98-02
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Escherichia coli
Rhodospirillum rubrum
Desulfobacterium anilini
Nitrospira marina
Thermodesulfobacterium hveragerdicum
Aquifex aeolicus
DR9-38-H3 clone group 6
Methanobrevibacter filiformis
Methanosphaera stadtmaniae
DR9-38-H3 clone group 7
Methanobacterium bryantii
Methanobacteriacaea
Methanobacterium subterraneum
DR9-38-H3 clone group 8
Methanobacterium defluvii
Methanobacterium wolfei
Methanobacterium thermoautotrophicum
Thermoplasma volcanium
Haloferax volcanii
Pyrococcus furiosus
Pyrobaculum neutrophilum
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.1
0
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• Key Observations
• Abiogenic hydrocarbos (e.g. methane)
• Methanogens present
• No major changes in anything over transect
• High acetate, H2, CH4
• Low SO4-2 (No S isotopes)

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Microbial community assessment of intact fracture
surfaces and pristine fissure waters from the
ultradeep continental subsurface. EV8-18 EB5/EB6
Moser, D.P., G. Wanger, B. Tipple, T.M.
Gihring, T.C. Onstott, J.K. Fredrickson, F.J.
Brockman, D.L. Balkwill, M.E. Dollhopf, S.
Pfiffner, L.M. Pratt, B. Sherwood Lollar, G.
Southam, L.H. Lin, M. Davidson, W. Seymor, C.
Ralston, Environmental Microbiology in
Preparation
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Location EV8-18 (gt 1 km into virgin rock)
1 km
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Fracture Surface from Core EB5
Fracture surface with intact silica infilling

• Service Water
• Drill Water
• Fissure water 1 hr
• Fissure water 48 hrs
• Fissure Water 30 days
• Fissure Water 70 days
• Fracture Surface
• Parings
• Inner Subsample

Note Sample was on dry ice within 10 minutes
of being removed from the Hole
SEM of fracture surface from work in progress by
Greg Wanger and Gordon Southam
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Bacterial and Archaeal T-RFLP Profiles
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Metals (ppm)
1 EV818EB5102502 (T 0) 2 EV818EB5102702 (T
2da) 3 EV818EB6102702 (T 30da?) 4
EV818EB6120702 (T ca. 70? da
Observations 1) Huge, quick drop in Fe and
Mn - Is the iron derived from iron filings
left over from the destruction of drill bits?
Where did it go? - Where did all that Mn
come from? Where did it go? - Its not in the
sheet yet, but there was a lot of NH4 initially
too.
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Nitrate from service water acting as a tracer
after water hit
Nitrate (complete series)
Conclusions Service and drill water high in
nitrate (as expected), after water intersect,
most of the nitrate is flushed out within 1 hr.
Nitrate is b.d. for remainder of time course.
50.000
Service Water
45.000
40.000
Drilling Water (Pre-Intersection)
35.000
Nitrate PPM
30.000
25.000
20.000
15.000
10.000
T 2 da
5.000
0.000
1
2
3
4
5
6
Water strike
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Archaeal and bacterial populations of an
ultradeep karstic aquifer. And Massive Filter
Methods Write-up? Moser, D.P., T.M. Gihring,
T.C. Onstott, J.K. Fredrickson, F.J. Brockman,
D.L. Balkwill, M.E. Dollhopf, G. Wanger, B.
Tipple, S. Pfiffner, L.M. Pratt, G. Southam, L.H.
Lin, M. Davidson. Journal of Cave and Karst
Studies, in preparation.
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DR9-IPC Archaea
uncultured archaeon SAGMA-K
SAVV137
Methanobrevibacter smithii
Methanobrevibacter cuticularis
SAVV145
SAF120
Methanobacterium bryantii
Methanobacterium curvum
Methanosphaera stadtmaniae
SAVV119
SAC174
hydrothermal vent clone pISA14
marine sediment clone SB-17a1A11
SAVV111
deep subsurface clone SAGMA-J
deep subsurface clone SAGMA-Q
deep subsurface clone SAGMA-I
SAP132
deep subsurface clone SAGMA-Y
deep subsurface clone SAGMA-2
uncultured archaeon
SAVV132
uncultured crenarchaeote
deep subsurface clone SAGMA-1
Acinetobacter junii
Planctomyces limnophilus
0.10
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DR9-IPC Firmicutes
SAVV3
SAVV39
Desulfotomaculum aeronauticum
Desulfotomaculum alkaliphilum
SAZ32
uncultured bacterium
uncultured bacterium
SAVV7
uncultured bacterium
Desulfotomaculum kuznetsovii
Peptococcus niger
Clostridium thermolacticum
SAVV41
Bacillus jeotgali
Bacillus infernus
Lactobacillus fermentum
Mycoplasma capricolum
SAVV27
SAVV10
uncultured bacterium, AY340841
SAD87
SAH5
uncultured bacterium, AF469402
Chlorobium vibrioforme
81E4IPC
uncultured Termite group 1 bacterium
SAB53
SAH44
SAVV1
SAVV48
uncultured bacterium, AY221048
Planctomyces maris
Planctomyces limnophilus
deep subsurface clone SAGMA-Y
Methanobacterium bryantii
0.10
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DR9-IPC Proteos

• No Thiothrix
• None in common between DR4IPC and DR9IPC

0.10
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Biogeographical Patterns of Bacteria and Archaea
in relation to Hydrogeological Variables the
Ultradeep Subsurface. Moser, D.P., T.M. Gihring,
T.C. Onstott, J.K. Fredrickson, F.J. Brockman,
D.L. Balkwill, M.E. Dollhopf, L.H. Lin, M.
Davidson. ??, in preparation.
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Biogeography1 (Widespread Desulfotomaculum-like
Organism)
West Drie 9 (Dream)
Evander 8-21
Evander 8-18 (EB6)
West Drie 9 (Dream)
SAZ7
Mponeng 104
SAC67
SAFF33
Beatrix 1-16
SAY1
MP12936
Mponeng 104
391-2-2
MP13120
West Drie 9 (Dream)
SAF7
uncultured bacterium, AF486685
uncultured bacterium, AF486697
Desulfotomaculum kuznetsovii
Desulfotomaculum australicum
East Drie 5 Shaft
uncultured bacterium, clone group17
uncultured bacterium, clone group18
Desulfotomaculum geothermicum
Desulfotomaculum alkaliphilum
Desulfotomaculum ruminis
Peptococcus niger
Clostridium thermolacticum
Lactobacillus fermentum
Bacillus infernus
Mycoplasma capricolum
Aquifex aeolicus
Thermodesulfobacterium hveragerdicum
Nitrospira marina
0.10
Red Clones Almost Identical set
Blue Clones South Africa
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Biogeography2 (SAGMA-12-like Archaea)
Evander 8-18 (EB6)
SAFF110
West Drie 6-38
uncultured archaeon SAGMA-12
Mponeg 104
MP110919
Evander 8-18 (EB6)
SAFF114
West Drie 6-38
uncultured archaeon SAGMA-15
West Drie 6-38-SC1
uncultured archaeon, AF486656
West Drie 6-38
uncultured archaeon SAGMA-13
SAB158
SAB130
Northam-27-Hose1
SAB133
deep subsurface clone SAGMA-H
deep subsurface clone SAGMA-U
West Drie 6-38 and Beatrix 2 Shaft
deep subsurface clone SAGMA-J
deep subsurface clone SAGMA-Q
deep subsurface clone SAGMA-G
Methanosarcina sp.VeA23
Methanocorpusculum parvum
"Cenarchaeum symbiosum"
Methanococcus voltae
Methanobacterium wolfei
"Pyrococcus abyssi"
Nanoarchaeum equitans
Thermodesulfobacterium thermophilum
Desulfotomaculum geothermicum
0.10
Red Clones Almost Identical set
Blue Clones South Africa
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• OBSERVATIONS
• DLO" and SAGMA-12 group found in 3 of the exact
  same holes
• EV818-EB6 (2002)
• MP104 (2002)
• W6-38-SC1 (1998)
• Close relatives of both scattered among West
  Drie, Evander and Beatrix
• Only mismatch is SAGMA-12 in Northam
• HYPOTHESES
• Wide distribution in modern time?
• Remant subsurface populations, long isolation,
  evolutionarily stable?
• Close relative of DLO, not exactly the same

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Occurances of possible SAGMA-12 in older West
Drie Samples. Note (SAGMA-12 has two possible
ribotypes due to single base chance in related
sequences
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DR504 Over Time From hole E546Bh1
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Is DR504 Indigenous?
55 oC
18 oC
28 oC
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Mponeng and 6Shaft West Drie red triangles
But What about Evander?
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Samples of Relevance Waters PLFA BactPCR
Arch PCR Service water taken from inlet
hose to drill rig on day of intersection Y? Y Y
Drilling rig water from EB5 taken several
hrs before water strike Y? Y Y Fissure
water from EB5 taken 1 hr after water
strike Y? Y neg so far Fissure water
from EB5 taken 2 days after water
strike Y? Y neg so far Fissure water
from EB6, surrigate for EB5 water 30 days post
water strike Y? Y Y Fissure water from
EB 6, surrigate for EB5 water 70 /- days post
water strike Y Y
Cores Actual fracture surfaces from
about 60 m Fracture surface ? Y Y Internal
from infilled mineral ? Y Y Note All
water samples complete gas and chemsitry
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BACKGROUND Evander 8-18 was sampled for
biology, gas and chem during attempt to sample
cores for porewater gases by Moser, Wanger and
Tipple in October of 2002, with considerable help
from Walter Seymore, 8 Shaft Geologist. Prospect
hole EB6 (450 m) was drilled about a month prior
to our major samplings on 10-25-02 and 10-27-02.
Hole EB5 intersected water at about 60 meters
depth on 10-25-02. Complete water sample sets
were taken from EB5 on 10-25 02 (1 hr post
intersection) and 2 days later on 10-27-02. Hole
EB6 was sampled on 10-27-02 and our hope was that
this sampling would be representative of what we
would have seen if we had been able to sample EB5
one month later (assuming both waters are from
the same fracture system). EB6 was also sampled
on 12-13-02 by Onstott et al. Together we are
hoping to use these samplings to roughly
approximate the aging of a borehole water
intersection beginning at 1 hr post intersection
out to about 70 days. Almost none of the data are
in yet, but it was an amazing sample set.
Methane and ethane from the 12-13-02 sampling
shows abiogenic (Kidd Creek type) hydrocarbon.
The two hole outlets are about 20 cm apart. Most
exciting, the drill rig was 1 km into virgin
rock. OBJECTIVES Monitor development of water
and gas chemistry and microbial community
structure over time in an effort to learn about
the aging of mine boreholes and get a sense for
how representative they are of the virgin deep
subsurface. After exhaustive study of known
sources of micro contamination, by exclusion,
look for microbes which are truly indigenous. In
the 1 hr sample, there will not have been
sufficient time for weeds to have grown. This is
our first opportunity of this type. If there are
major lineages in the 1 hr sample that are not in
the service water or drilling mud, I will be
content to regard them as most likely indigenous.
If the EB5-10-25-02 population is very similar to
drilling mud, I will be most inclined to think
the fissure was sterile (or had cell numbers
below whatever our limit of detection turns out
to be). Anyone willing to wager? DNA libraries in
progress now.
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Physical Properties
1 EV818EB5102502 (T 0) 2 EV818EB5102702 (T
2da) 3 EV818EB6102702 (T 30da?) 4
EV818EB6120702 (T ca. 70? da
Observations 1) Eh Goes up slightly 2)
Temperature goes up slightly? -Probably
misleading since EB6 (samples 3 and 4) is a
deeper hole with two water intersections. Maybe
the deeper one is hotter? These dates are
guesses
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1 EV818EB5102502 (T 0) 2 EV818EB5102702 (T
2da) 3 EV818EB6102702 (T 30da?) 4
EV818EB6120702 (T ca. 70? da
DIC, Formate, Acetate (ppm)
Observations 1) Quick formate spike,
followed by transient acetate spike - Is
there a way to make formate abiotically? -
Acetogenesis kicks in and then declines?

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Cations (ppm)
1 EV818EB5102502 (T 0) 2 EV818EB5102702 (T
2da) 3 EV818EB6102702 (T 30da?) 4
EV818EB6120702 (T ca. 70? da
Observations 1) Upward Trends Na, Ca,
maybe Ca 2) Downward Trends K, Mg2, Ba2
- What kind of weathering might account for
this?
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Sulfur cpds (ppm)
1 EV818EB5102502 (T 0) 2 EV818EB5102702 (T
2da) 3 EV818EB6102702 (T 30da?) 4
EV818EB6120702 (T ca. 70? da
Observations 1) Increases in both sulfide
and sulfate - Not exactly mass balance
here - Maybe isotopes will help
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Anions (ppm)
1 EV818EB5102502 (T 0) 2 EV818EB5102702 (T
2da) 3 EV818EB6102702 (T 30da?) 4
EV818EB6120702 (T ca. 70? da
Observations 1) Cl-, Br- unchanged 2) F'
increases 3) T 1 hr is contaminated with
service water (about
180 Service water/fissure water)

• Where did that F- come
• from?

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Proposed Analyses (what else?) Waters DNA
(in progress) Gihring, Moser et
al. Cultivations (read on) Gihring,
Moser? PLFA (Susan?) Pfiffner et al. Aq
Chemistry (most done) Onstott et al. S
isotopes Pratt, Boice, etc. Gases and
hydrocarbon chem/isotopes Sherwood Lollar et
al. Radiogenic Isotopes for Age of
waters? Lippmann Rocks DNA Moser,
Gihring et al. PLFA (send to Susan, if willing
after processing) Pfiffner et al. Silver foil,
SRB Activity Davidson and others from Onstott
lab EM showing fracture surfaces Southam,
Wanger, Lemke, Davidson EM looking for
microfossils in fracture filled material?
Southam, Wanger, Lemke, Davidson Crush and
leach experiment for porewater contents? Onstott
et al.
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• PLAN OF ACTION (PNNL Action items)
• Finish water sample bacterial 16S clone libraries
• Keep working on getting 16S Archaeal PCR where we
  dont have them
• Try Functional Primers?
• Analyze all the clones
• Other Analyses we really want
• Sulfur isotopes
• More hydrocarbon isotopes
• Age of the water
• An explanation for the high iron initially

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A K441FW156HWDHole1 100201 5L B NO27RWFWDEHose1
100501 4L C EV8.21FW101601 101601 5L D DR546BH
1 103001 4L E kit control/blank F DR938H3 Om
packered 110701 7L G K441FW156Hole3 111401 3.5
L H EV221BH2 111601 0.25L I BE341FW 032701 4
.8L J BE339FW 032701 4.5L K BE16FW 031401 3L
L-03 DR9IPC H1 101602 M-03 DR938CH1 102102
N-03 DR938CH1 111702 O-03 DR938H3 111702 P-0
3 EV818SW 102502 Q-03 EV818BHEB5
DRLW 102502 R-03 EV818BHEB5 102502 S-03 EV818B
HEB5 102702 T-03 DR938H3-SW 102401 U-03 DR938H
3-0meters 102501 V-03 DR938H3-125meters 102501
W-03 DR938H3-250meters 102501 X-03 DR938H3-390m
eters 102501 Y-03 DR938H3-550meters 102501 Z-0
3 DR938H3-648meters 102501 AA-03 MP104XC64BH 1
10902 BB-03 DR938H3-BF 102401 CC-03 DR5E5030Xcu
t acidseep 101502 DD-03 DR5E5030Xcut
stopewater 101502 EE-03 DR5E50SW 101502 FF-03
EV818BHEB6 102702 GG-03 EV818BHEB6 111502 HH-0
3 EV818 CoreEB5 Fracture Surface 102502 II-03 EV8
18 Core EB5 Inner Subsample 102502 JJ-03 EV818
Core EB5 Parings 102502 KK-03 MP104 XC64
092702 LL-03 DR940 SW 092602 MM-03 MP104
091902 NN-03 MP104 091602 OO-03 EV818BHEB5
2 102502 PP-03 EV818BHEB52 102702 QQ-03 DR9I
PCBH1 (Mini-Massive) 101602 RR-03 EV818SW
SS-03 E546BH1 101802 TT-03 Field Lab
Sink UU-03 Filter GIT control VV-03 DR9IPC
(Massive Filter) 101602

• 48 Samples
• Literally including the kitchen sink