FEMS Microbiology Reviews 20 (1997) 399^414
Microbial life in deep granitic rock
Karsten Pedersen *
Institute of Cell and Molecular Biology, Department of General and Marine Microbiology, Go
ë teborg University,
Medicinaregatan 9C, S 413 90 Go
ë teborg, Sweden
Abstract
Granitic rock has aquifers that run through faults and single or multiple fracture systems. They can orientate any way,
vertically or horizontally and usually, only parts of hard rock fractures are water conducting. The remaining parts are filled by
coatings of precipitated minerals, and clay and gouge material. Sampling hard rock is difficult and the risk of contamination
due to intrusion of drilling fluids and cuttings in aquifers is obvious. A recent investigation of the potential for contamination
of boreholes in granite during drilling operations, using molecular and growth methods, showed that predominating
microorganisms in the drilling equipment were absent in groundwater from the drilled boreholes. The total number of bacteria
found in subterranean granitic environments ranges from 10 up to 10 cells per ml groundwater, but the number of cultivable
microorganisms is usually much lower. We have used culturing techniques with numeric taxonomy for the identification of
cultivable microorganisms and the 16S rRNA gene technique to determine bacterial diversity in granitic groundwater.
Members of the genera
,
,
and
have been found. Several biogeochemical processes in granitic rock have been demonstrated where microorganisms
seem to be of major importance. One process is the mobilization of solid phase ferric iron oxy-hydroxides to liquid phase
ferrous iron by iron reducing bacteria with organic carbon as electron donor. Another biogeochemical process found to be
important is the reduction of sulfate to sulfide by sulfate reducing bacteria. They frequently appear in granitic aquifers at
depths, and seem to prefer a moderate salinity, approximately 1%. When groundwater rich in ferrous iron, manganese(II) and
reduced sulfur compounds reaches an oxygenated atmosphere such as an open tunnel, gradients suitable for chemolithotrophic
bacteria develop. A third process is the conversion of carbon dioxide to organic material with hydrogen as the source of energy,
possibly formed through radiolysis, mineral reactions or by volcanic activity. Recent results show that autotrophic
methanogens, acetogenic bacteria and acetoclastic methanogens all are present and active in deep granitic rock. These
observations announce the existence of a hydrogen driven deep biosphere in crystalline bedrock that is independent of
photosynthesis. If this hypothesis is true, life may have been present and active deep down in the earth for a very long time, and
it cannot be excluded that the place for the origin of life was a deep subterranean igneous rock environment (probably hot with
a high pressure) rather than a surface environment.
3
7
Bacillus, Desulfovibrio, Desulfomicrobium, Eubacterium Methanomicrobium, Pseudomonas Serratia
Shewanella
Keywords :
16S rRNA; Deep biosphere; Granite; Microorganism; Aëspoë Hard Rock Laboratory
* Tel.: +46 (31) 773 2578; Fax: +46 (31) 773 2599; E-mail: [email protected]
0168-6445/97/$32.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
S0168-6445(97)00022-3
PII
400
K. Pedersen / FEMS Microbiology Reviews 20 (1997) 399^414
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Geology, hydrology and geochemistry of Swedish crystalline bedrock
2.1. Geology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Hydrology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Geochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Getting access to the deep granitic environment . . . . . . . . . . . . . . . .
3.1. Drilling and tunnelling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Culturing and molecular control for contamination . . . . . . . . . .
4. Life in hard rock tunnels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Diversity and distribution of bacteria in granitic groundwater . . . . . .
5.1. Culturing methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Sequencing 16S rRNA genes . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3. In situ hybridization with group speci¢c nucleic acid probes . . .
6. Biogeochemical processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1. Iron reducing bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2. Sulfate reducing bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7. Hydrogen-dependent microorganisms . . . . . . . . . . . . . . . . . . . . . . . .
7.1. Hydrogen and methane in deep groundwater . . . . . . . . . . . . . . .
7.2. Acetogenic bacteria in deep granitic groundwater . . . . . . . . . . .
7.3. Methanogens in deep granitic groundwater . . . . . . . . . . . . . . . .
8. Conclusions ^ The deep hydrogen driven biosphere . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
In recent years, published papers on various aspects regarding the microbiology of subterranean environments have increased in numbers. There has
been a signi¢cant expansion in the understanding
of the bacterial ecology of shallow groundwater systems down to some 50^100 m, accurately reviewed
by Ghiorse and Wilson [1] and Matthess et al. [2],
and our knowledge is currently increasing about environments deeper down into the crust of the earth
[3^6]. Deep subsurface environments vary considerably in composition, from soft sandstone and hardened sedimentary rocks to very hard igneous rock
types. The main purpose of this review is to present
hypotheses, theories and results on microbial life in
one of the hardest and most common rock types of
the earth crust, granite.
The Swedish research program on subterranean
microbiology [5,7,8] has been performed on two
sites, the Stripa research mine in the middle of Sweë spoë area, next to the Baltic sea in the
den and the A
southeastern part of Sweden. The Stripa mine is sit-
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400
401
401
401
402
404
404
405
406
407
407
407
408
410
410
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411
411
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412
412
413
413
uated 250 km west of Stockholm and was an iron
mine until 1976. A total of 16.5 106 tons of iron ore
has been mined out since 1448. The ore consisted of
a quartz-banded hematite and occurred in a lepatite
formation. Adjacent to the lepatite is a large body of
1.7 billion year old medium-grained granite, in which
the Stripa project experiments have been performed.
The mine was used as a deep underground research
ë spoë infacility between 1976 and 1994 [9^11]. The A
vestigation area, situated on the south-east coast of
Sweden, is a part of the Precambrian bedrock in
southeastern Sweden where the Smaîland granites
predominate the older, Sveocokarelian complexes.
ë spoë Hard Rock Laboratory
This is where the A
(HRL) is situated, at 460 m below the surface of
ë spoë (Fig. 1). The A
ë spoë HRL has been
the island A
constructed as a part of the Swedish nuclear waste
disposal program and the work has been divided into
three phases: the pre-investigation (1986^1990), the
construction (1990^1995) and the operating (1995^)
phases. Microbial investigations have been performed during all three phases [12^15] and the research is currently continuing.
U
K. Pedersen / FEMS Microbiology Reviews 20 (1997) 399^414
401
Fig. 1. A: The sampling situation at the Aëspoë Hard Rock Laboratory in June 1996. The sample sites are depicted with their respective
borehole names. These names show the type of drilling (HBH, HA = percussion drilled; KAS, SA = core drilled), the tunnel length where
they were drilled and whether they were drilled on the left (A) or the right (B) side of the tunnel when going down. Major fracture zones
are marked with dashed lines and with their given names, generally indicating their geographic orientation. Possible £ow directions of
groundwater are indicated with arrows and the estimated in£ow rates of groundwater via the fractures to the tunnel are shown in brackets as l s31 . B: A fracture zone (RZ) with boreholes that were drilled in order to follow shallow groundwater intrusion through this major fracture zone into the tunnel. A side vault was constructed (not shown) from which the boreholes KR0012, 13 and 15 were drilled
perpendicular through the zone. Note that these three boreholes all sampled at 68 m below sea level (not shown).
2. Geology, hydrology and geochemistry of
Swedish crystalline bedrock
2.1. Geology
Sweden is part of the 1.6^3.1 billion years old
Fennoscandian Shield and a number of places
in Sweden have been investigated as study sites
for the deep disposal of spent nuclear fuel. The
crystalline rock considered has generally been of
granitic composition with quartz, feldspars and
mica as the bulk rock minerals. In addition to
that, there are accessory minerals which in£uence the hydrochemical conditions such as calcite
23
(pH and HCO3
3 ), pyrite (redox), apatite (HPO4 ),
3
£uorite (F ) and clay minerals (ion exchange).
Many of these occur as fracture ¢lling minerals
and some of them have been formed as a result
of weathering reactions. Minor amounts of iron(III)
oxy-hydroxy minerals are found in the fractures,
especially in the shallow ( 6 100 m) part of the
rock.
2.2. Hydrology
The distribution of £ow has an in£uence on
groundwater composition. The hydraulic conductivity varies considerably between di¡erent locations in
the rock, and structures like fracture zones may act
as conductors and have a dominating in£uence. Vertical conductive zones are important for groundwater recharge at depth. Horizontal zones may act
as hydraulic shields and separate groundwater with
di¡erent composition. Especially deep groundwater
with a relatively high salinity will have a higher density which helps to stabilize the layering. An example
of that has been studied in Finnsjoën [16]. The openings in rock fractures are potential channels for
groundwater. Model studies have been made on
£ow and transport in fractures with variable apertures [17]. The results suggested that considerable
channelling is to be expected in such fractures and
that there is a tendency for some pathways to carry
much more water than others. In a limited mass of
rock, one or a few channels will dominate £ow and
K. Pedersen / FEMS Microbiology Reviews 20 (1997) 399^414
402
transport of nutrients and microorganisms. Hydraul-
porous medium, the water £ow into the tunnel can
ic conductivities have been measured in boreholes at
still be illustrated by this £ow model. The £ow lines
di¡erent depths and this information together with
in Fig. 2 are regular because of the assumed homo-
the groundwater surface topography, which in Swe-
geneity. In reality the £ow path will be irregular, but
den is approximately the ground surface topography,
on a scale greater than some 50 m the £ow lines
is
¢eld.
will most probably have approximately the same
Groundwater £ow at some 500 m depth is calculated
2
1
year
[7]. Hyto be in the range of 0.01^1 l m
pattern as in the simpli¢ed model. Note that a sig-
draulic conductivity and £ow increase near the sur-
are situated deeper than the tunnel position consid-
face. At or below sea level the hydraulic gradient is
ered.
used
to
calculate
the
groundwater
3
£ow
3
ni¢cant part of the in£ow comes from aquifers that
evened out and therefore the £ow rate is very small
2.3. Geochemistry
there.
The hydraulic gradients increase considerably in
the vicinity of a tunnel causing a di¡erent £ow pattern
compared
to
before
tunnelling,
when
Groundwater under land in Sweden has in general
the
a meteoric origin. The in¢ltrating water is almost
groundwater £ow is small due to the small gradients
`pure water' from rain or melting snow with dis-
caused by natural water levels and the hydraulic
solved air as an important component. The processes
head distribution at deeper levels. Fig. 2 shows the
in the biologically active soil zone are therefore very
£ow pattern and hydraulic head distribution of a
important for the composition of recharge water.
ë spo
hypothetical homogeneous case for the A
ë HRL
Oxygen will be consumed and carbon dioxide added.
tunnel at 200 m below sea level (Fig. 1). No hydraul-
The carbonic acid will react with minerals such as
ic resistance was assumed around the tunnel. Even
calcite and feldspars and form carbonate ions and
though the rock is de¢nitely not a homogeneous
release calcium and alkali ions to the water. Ion ex-
ë spo
Fig. 2. Calculated hydraulic head distribution and £ow lines around the A
ë HRL tunnel. Modelled hydraulic head distribution (m) is
shown as isobars around the tunnel when it passes 200 m below sea level. No hydraulic resistance around the tunnel is assumed. The
£ow lines for particle traces (backtracked from the tunnel) are evenly distributed around the tunnel.
K. Pedersen / FEMS Microbiology Reviews 20 (1997) 399^414
change with clay minerals may a¡ect the proportions
between cations. Organic materials such as humic
and fulvic acids and other substances will be added
to the water from the soil. The biological processes
will also have a similar in£uence if seawater in¢ltrates through organic rich sea sediments.
At great depths or under the sea bottom, saline
water is found where chloride is the dominating
anion. The most common cation in saline groundwater is either sodium or calcium. The saline water
may have a marine origin but other end members are
also possible, depending on location and other conditions. Very deep, at depths of 1000^1500 m or
more, the salinity can be very high and reach well
above ocean seawater and even approach brine composition, i.e., 10% or more. It is also common that
saline groundwater is found at shallower depth in
coastal regions than further inland. This may of
course be relict seawater that in¢ltrated several thousands of years ago, when land near the coast in Sweden was covered by the sea due to the glacial depres-
403
sion (land pressed down by the ice cover). The
in¢ltration of seawater continued until land was reclaimed by the land uplift, which is still continuing in
Sweden. However, an alternative explanation can be
found in the lack of driving hydraulic force under
the `£at' surface of the sea. With no, or very low,
hydraulic gradient in the groundwater beneath the
sea bottom, fossil saline conditions can be preserved
for very long time periods and it must not always be
the result of a relatively recent in¢ltration of seawater. In other words, saline water may have originated even far before the last glaciation some 10 000
years ago.
Some typical groundwater compositions to be expected at di¡erent depths and locations encountered
in the course of research and exploration within the
Swedish radioactive waste management program are
given in Table 1. It is obvious that major constituents such as the cations sodium and calcium and the
anions bicarbonate and chloride can vary considerably in concentration depending on where and at
Table 1
ë spoë [44]
Chemical parameters of granitic Swedish ground water from boreholes in Finnsjoën [42], Klipperaîs [43] and A
Finnsjoën
Borehole
KFI09
KFI09
94
360
7.3
7.6
Depth
m
pH
mV
Eh
mg l31
Na‡
mg l31
K‡
mg l31
Ca2‡
mg l31
Mg2‡
mg l31
Sr2‡
mg l31
Fe2‡
Mn2‡
HCO3
3
F3
Cl3
mg l31
SO243
mg l31
HS3
mg l31
(N)
(N)
4 (N)
HPO243 (P)
SiO2 (Si)
U
mg l31
mg l31
I3
TOC
mg l31
mg l31
Br3
NO3
2
NO3
3
NH‡
mg l31
mg l31
mg l31
mg l31
mg l31
mg l31
mg l31
Wg l31
3245
ë spoë
A
Klipperaîs
Area
^
KKl01
KKl09
KAS03
KAS03
404
581
129
860
8.3
7.6
8.0
8.0
3300
3270
3270
3250
415
1500
47
15
600
3050
6
7
1
1
2
7
115
1700
14
29
162
4400
16
84
2
3
20
50
^
^
^
^
3
75
0.56
0.34
0.01
0.09
0.12
0.08
0.19
0.36
^
^
0.10
0.20
285
32
80
120
61
11
3
9
4
3
2
2
680
5200
45
6
1230
12 300
2
27
0.4
0.05
5
85
0.01
0.07
0.008
0.002
0.10
0.70
175
340
1.5
4.3
32
720
0.22
0.03
0.10
0.01
0.70
1.10
^
^
^
^
0.001
0.001
0.02
0.01
^
^
0.01
0.01
^
^
^
^
0.04
0.01
0.001
0.004
0.001
0.003
0.002
0.002
7.6
7.6
4.4
9.9
4.8
4.2
18
1.0
3.7
1.2
2.0
0.5
2.1
8.2
0.01
0.04
0.15
0.13
404
K. Pedersen / FEMS Microbiology Reviews 20 (1997) 399^414
Table 2
The content of nitrogen, hydrogen, helium and carbon containing gases, and the total volumes of gas extracted from
the Stripa borehole V2, the Laxemar borehole KLX01 and the Aëspoë boreholes KR0012, 13 and 15 [8,9,13]
Borehole
Sampling
N2
H2 +He
CO
CO2
CH4
C2 H 6
C2 H2ÿ4
(W l l 3 1 )
depth (m)
(W l l 3 1 )
(W l l 3 1 )
(Wl l31 )
(Wl l31 )
(Wl l31 )
(Wl l31 )a
Stripa
V2
799^807
25 000
6 10
61
32
245
0.3
6 0.1
V2
812^821
31 000
6 10
61
11
170
0.6
6 0.1
V2
970^1240
24 500
6 10
61
10
290
2.9
6 0.1
Laxemar
KLX01
KLX01
KLX01
groundwater from
Volume of
extracted gas (%)
2.4
3.4
2.7
46 500
37 000
18 000
4600
3500
2450
0.5
0.1
0.7
460
500
1 600
26
27
31
6 0.1
6 0.1
6 0.1
6 0.1
6 0.1
6 0.1
5.7
4.4
3.5
Aëspoë
KR0012
68
22 000
KR0013
68
25 000
KR0015
68
22 000
a The content of C H +C H .
2 2
2 4
40
110
64
0.1
0.2
0.1
6 050
9 640
15 037
1030
1970
4070
6 0.1
6 0.1
6 0.1
0.1
0.1
0.1
2.9
3.7
4.0
830^841
910^921
999^1078
which depth the samples have been taken. Chloride
behaves conservatively but many other ions obviously interact with the minerals. This is particularly
evident in groundwater with a marine origin. An
example of that is the ion exchange of calcium for
sodium and vice versa. A further observation is that
ions like potassium and magnesium, which are common in seawater, are evidently suppressed in groundwater ^ presumably by reactions with the minerals.
Even sulfate is partly consumed, probably by sulfate
reducing bacteria. Carbonate is less common at
depth. Possible explanations are that slow reactions
with the rock minerals cause precipitation of carbonate as calcite and autotrophic microbial organic carbon and methane formation.
The pH of granitic groundwater in Sweden is buffered by the carbonate system. Calcite is abundant as
mineral and feldspars can also react with acids.
Therefore `acid rain' or any similar disturbance of
pH does not propagate very far down underground.
Deep groundwater does not contain any oxygen.
Measurements of redox potential with Eh electrodes
give values between 3100 and 3400 mV. There is a
dependence of Eh on pH and Fe2‡ concentration but
the low concentrations of redox active species in
groundwater make the measurement of Eh a delicate
operation. In situ measurement has been found to
o¡er the best quality [18]. The low content of for
example Fe2‡ gives the water only a low redox bu¡er
capacity. However, a considerable capacity is contained in the rock and its content of iron(II) minerals
and pyrites [19]. Groundwater contains dissolved
gases such as nitrogen, carbon dioxide, methane, hydrogen, helium (Table 2), neon, argon, krypton and
radon. Oxygen is only found at relatively shallow
depths.
3. Getting access to the deep granitic environment
3.1. Drilling and tunnelling
All sampling of subterranean environments requires substantial e¡orts in drilling or tunnelling.
The possibility of microbial contamination of the
sampled specimens by access operations is indisputable and must be considered when interpreting the
obtained results. Conditions like the geological formation, the history of a borehole or a tunnel, available equipment and the type of sample considered
are variables that will in£uence the prospect of getting non-contaminated samples. Depending on these
prevailing conditions, realizable precautions against
contamination vary from virtually none to speci¢c
devices aimed at sterile sampling [4]. Coring crystalline bedrock requires vigorous drilling action with
high drilling £uid pressures. The risk of microbial
contamination of the aquifers with the drill water
405
K. Pedersen / FEMS Microbiology Reviews 20 (1997) 399^414
used to transport the drill cuttings out of the borehole during drilling is obvious. Some di¡erent measures can be applied to reduce such contamination.
Clean drill water as free from microbes as possible is
an essential prerequisite. Pumping of a borehole to
measure its maximum hydraulic water capacity is
often done and will concurrently clean the aquifers
and the borehole from drill water, mud and cuttings.
In addition, a control of the mixing of drill water
in the groundwater can be made by introducing
di¡erent tracers in the drill water that can subsequently be analyzed for in the groundwater samples
[4]. The necessity of clean drilling equipment free
from contaminations is evident, but not always
achievable.
viewed by Amann et al. [20]. Non-culturing techniques such as extraction and sequencing of the
16S rRNA gene can be applied to overcome that
problem [5,10,15]. Recently, culturing and molecular
techniques were used to investigate possible and lasting contamination of boreholes drilled in crystalline
bedrock at the `SELECT' site in the Aëspoë HRL, at
depths of 300^440 m (Fig. 1). Samples were collected
from the drill water, the drilling equipment and from
the drilled boreholes and analyzed. Total numbers of
bacteria, viable aerobic and anaerobic plate counts
and most probable numbers (MPN) of sulfate reducing bacteria (SRB) were performed parallel with the
analysis of 16S rRNA gene diversity of the samples
(Table 3).
The measures taken to avoid contamination of the
boreholes included steam cleaning of all temporary
and permanent equipment with a hot water high
pressure cleaner. Using deep groundwater from borehole HD0025 at site level as drill water (Fig. 1) excluded the possibility of introducing contaminating
microorganisms with surface originated drill water.
This possibility to use surface water as drill water
3.2. Culturing and molecular control for
contamination
The use of culturing methods alone for the control
of potential contamination of boreholes is not
enough due to the well documented `great plate
count anomaly' of environmental samples as re-
Table 3
Drilling and sampling schedule and counts of microorganisms in groundwater from newly drilled boreholes and the drilling equipment
Sampled
site
Section
sampled
(m)
Sampling
date
Drilled
borehole
16S rDNA
Total number of Aerobic viable
extraction and bacteria ml31
count of bacteria
sequencing
105 (S.D.)
ml31 (S.D.)
Anaerobic viable
count of bacteria
ml31 (S.D.)
MPN SRB
ml31
HD0025A
HD0025A
HD0025A
Tubing
Drill water
containera
Drill water
containera
Drilling
machinea
KA2858A
KA2858A
KA3005A
KA3105A
KA3105A
KA3105A
KA3105A
0^17.0
0^17.0
0^17.0
n.r.d
n.r.
94/11/30
94/12/14
95/01/17
95/01/17
94/12/14
KA3005A
KA3105A
KA2858A
KA2858A
KA3105A
yes
no
yes
yes
yes
0.806 (0.20)
0.851 (0.12)
ic
22.3 (0.60)
1.030 (0.81)
80 (61)
^b
70 (28)
s 100 000
44 500 (22 700)
75 (34)
^
60 (14)
s 100 000
1 930 (808)
100
^
100
100
10
n.r.
95/01/17
KA2858A
yes
4.34 (0.33)
7 400
3 700
10
n.r.
95/01/17
KA2858A
yes
3.94 (0.25)
6 600
3 100
100
0^59.7
39.8^40.8
0^58.1
0^70
17.0^19.5
17.0^19.5
22.5^24.5
95/02/02
96/06/27
94/12/14
95/01/17
96/02/02
96/06/27
96/06/27
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
yes
yes
no
yes
no
yes
yes
0.12 (0.007)
^
0.210 (0.01)
0.30 (0.01)
0.92 (^)
^
^
^
125
9 (8)
13 (10)
^
^
^
^
^
10 (10)
62
^
^
^
10
^
0
U
Borehole HD0025A (Fig. 1) was used as a source of drill water during drilling of the other boreholes.
S.D., standard deviation.
b
No data.
c
Impossible to count due to background £uorescent precipitates.
d
Not relevant.
a
s 1 000
10 000
^
^
406
K. Pedersen / FEMS Microbiology Reviews 20 (1997) 399^414
was considered during the planning process but was
abandoned due to the obvious risk of introducing
oxidized water into the rock aquifers. Part of the
SELECT site will be used for studies on the reducing
capacity of fracture minerals and oxygenated water
would have spoiled that series of experiments. An
obvious source of microbial contamination was the
tubing taking drill water from HD0025A to the drill
water container as can be judged from signi¢cantly
elevated total and viable counts in samples from the
tubing compared to the other sampled sites (Table
3). This tubing was a £exible, reinforced rubber tube
of a type that could withstand the signi¢cant pressure of groundwater in HD0025A, approximately
40 atmospheres. The distances between this borehole
and the drilled boreholes KA2558A and KA3105A
(Fig. 1) were 642 m and 95 m respectively, which
required at least the same tube lengths. Rubber tubing material contains organic component such as
softeners and stabilizers which slowly leak out to
the water in the tubing. Therefore, such tubing is
susceptible to bio¢lm formation of microorganisms
that can grow with these compounds [21,22]. Cleaning e¡orts will not be lasting (unless the tubing is
totally sterilized, which was impossible) as new bio¢lms will develop as soon as water enters the tubing
again.
The source of microbial contamination from the
tubing o¡ered an excellent possibility to evaluate
whether large numbers of certain species in drilling
equipment will cause lasting contamination of a
borehole with the same species. The culturing technique could not detect such contamination of the
drilled boreholes (Table 3). A similar result was obtained when the diversity of 16S rRNA genes in the
samples was compared (unpublished results, not
shown). Two strains of the genus Shewanella were
indicated to be dominating in the drill water and in
HD0025, but they could not be detected in water
from the drilled boreholes. In conclusion, it could
not be proved that microorganisms in the drilling
equipment contaminated the drilled boreholes. This
is an expected result, because the growth conditions
are di¡erent in the rock compared to the drilling
equipment. Microorganisms that are adapted to
grow on the nutritious tube walls would probably
not survive in the nutrient poor rock environment
and vice versa.
4. Life in hard rock tunnels
Excavation for tunnels, mining etc. introduces several changes into the subterranean environment that
will induce activities in the tunnel by microorganisms
other than those present in the fractured rock. Oxygen is normally introduced into tunnels by ventilation which makes growth of aerobic bacteria possible. As the groundwater at depth is usually anoxic
with a low redox potential (Tables 1 and 2), marked
redox and oxygen gradients will develop when such
groundwater reaches the oxygenated tunnel atmosphere. Typical redox pairs participating in these gradients are manganese(II) oxidizing to manganese(IV), ferrous iron to ferric iron, sul¢de to
sulfate (Table 1) and probably also methane to carbon dioxide (Table 2). Such gradients are the habitats for many di¡erent lithotrophic and also heterotrophic bacteria. Among them are the iron,
manganese, sulfur and methane oxidizing bacteria
that generate chemical energy for anabolic reactions
through the oxidation of reduced inorganic compounds and methane with oxygen. The energy gained
by the lithotrophs is used to reduce carbon from
CO2 to organic carbon and this is the ¢rst step in
an environmental succession that eventually ends as
a reduced environment again.
Commonly, seeps of groundwater from fractures
intersected by the Aëspoë tunnel or £ows of groundwater from boreholes turn light brown to dark
brown from precipitates that sometimes can be
very voluminous. They usually appear within some
weeks after excavation/drilling and have in some
cases reached a thickness of 10 cm or more. The
most frequently occurring inhabitant in these precipitates is the lithotrophic iron oxidizing bacterium Gallionella ferruginea [23^26]. It forms moss
like covers on rocks and sediments in ponds in
the tunnel and is very abundant close to the out£ow of groundwater from rock wall fractures [7].
At many such out£ows, white, threadlike structures are observed. Microscopic observation has revealed them to be sulfur oxidizing bacteria of di¡erent types, both extracellular and intracellular
deposition of sulfur has been observed. Especially
tunnel sections below the sea bed with ongoing sulfate reduction harbor this type of bacteria [7]. Sequencing the 16S rRNA gene from one of these sites
K. Pedersen / FEMS Microbiology Reviews 20 (1997) 399^414
has indicated the genus
published).
Thiotrix to be present (not
5. Diversity and distribution of bacteria in
granitic groundwater
5.1. Culturing methods
Total numbers of microorganisms in subsurface
granitic environments range from some 103 up to
107 cells ml31 [5]. We have used culturing techniques
with numeric taxonomy for the phenotypic characterization and the 16S rRNA technique for genotype
407
characterization to determine bacterial diversity in
granitic groundwater. Facultatively anaerobic, heterotrophic bacteria were identi¢ed from boreholes
KAS02 and 03 during the pre-investigation phase
ë spoë as belonging to the genera Pseudomonas
at A
and Shewanella [12]. Later, identi¢cation of heterotrophic facultative bacteria from the Aëspoë tunnel
demonstrated that members of the Serratia, Bacillus,
Desulfovibrio, Desulfomicrobium, Eubacterium and
Methanomicrobium genera are also present [15,27].
5.2. Sequencing 16S rRNA genes
The ¢nding of many new and unknown bacterial
ë spoë HRL tunnel.
Fig. 3. Evolutionary distance tree based on the 16S rRNA gene sequences of clones from di¡erent boreholes in the A
Major phylogenetic groups of bacteria have been designated with their generally accepted names. As references, some 16S rRNA gene sequences of known bacteria from the EMBL database have been added to the tree and are indicated with their Latin names. The branch
lengths are proportional to calculated evolutionary distances.
408
K. Pedersen / FEMS Microbiology Reviews 20 (1997) 399^414
16S rRNA sequences in natural environments is a
commonly reported result [10,28^30]. This was also
ë spoë HRL
the case when 16S rRNA genes from the A
tunnel boreholes were compared with DNA databases [15]. There is not any accepted value of the
percent identity at which two 16S rRNA genes can
be concluded to belong to the same genus or species.
It can be quite di¡erent for di¡erent genera [31] and
is also due to whether total or partial 16S rRNA
genes are compared. It has been suggested, based
on a comparison of rRNA sequences and on
DNA-DNA reassociation, that a relation at the species level does not exist at less than 97.5% identity in
the 16S rRNA sequence. At higher identity values,
species identity must be con¢rmed by DNA-DNA
hybridization [32]. Fig. 3 shows a phylogenetic tree
ë spoë clone group sequences. Six distinct
for 48 A
groups of phylogenetically related bacteria were
found [33], the alpha, beta, gamma, delta and epsilon
groups of the Proteobacteria, and Gram-positive
bacteria. The remaining sequences were only very
distantly related to known, named and sequenced
bacteria reported to the databases. Accepting the
level of 97.5% conservatively, as identifying a sequence approximately at the genus level, some conclusions can be drawn about the sequences from the
ë spoë granitic groundwater. The Bacillus (A5g,
A
98.6%), Desulfovibrio (A6-7hq, 97.7%) and Acinetobacter (A24optmn, 98.6%) like sequences had identities higher than 97.5% with 16S rRNA sequences in
the database, and may be regarded as identi¢ed at
the genus level. One of the clone groups could be
identi¢ed as a member of the domain Eukarya,
a yeast, Saccharomyces (A61upm, 97.6%) [15].
The only isolate whose sequence was also found
in the clone libraries (clone A1ghq) was Aspo-4.
The 16S rRNA of Aspo-4 showed 91.7% identity
with the Gram-positive bacterium Eubacterium limosum, which is too low for identi¢cation. However,
preliminary phenotypic characterization indicates
this isolate to be a homoacetogenic species. It
was isolated from SA813B (Fig. 1) and its 16S
rRNA sequence was found in groundwater and surfaces from this borehole, from KR0013 groundwater
and from several of the SELECT boreholes (300^
440 m).
When PCR ampli¢cation is used for the determination of species diversity, the result may be biased
due to methodological problems, such as uneven extraction of DNA and biased PCR due to di¡erences
in genome size [34]. One of the most important
causes of bias is that organisms belonging to the
domain Archaea have only one or a few gene copies
of the 16S rRNA gene while bacteria can have several copies, 5^7 or more, and this will bias PCR
ampli¢cation towards bacteria [20,29]. Therefore, using PCR primers that are speci¢c for archaean 16S
rRNA gene sequences in parallel with universally
conserved ones will enhance the detection of microorganisms belonging to the domain Archaea. The
results presented in Fig. 3 were obtained using the
universal primers only and should therefore be expected to reveal mainly bacterial diversity and distribution.
5.3. In situ hybridization with group speci¢c nucleic
acid probes
The inability of our universal primers to detect
members of the domain Archaea led us to use nucleic
C
Fig. 4. A: Granite rock coupons were exposed overnight to a growing culture of Shewanella putrefaciens and washed with a bu¡er. Subsequent in situ hybridization with a Cy-5 labelled probe for the domain Bacteria (EUB-338) revealed attached bacteria on the surface. The
Cy dyes are based on the cyanine £uor and all seven di¡erent £uors o¡er intense colors with narrow emission spectra (Amersham Life
Science). A Molecular Dynamics 2010 confocal laser scanning microscope equipped with a Kr/Ar laser was used for observation with the
software Image Space running on a Silicon Graphics UNIX based computer. The hybridization signal obtained was maximal with virtually no background at all, as can be seen from the intensity diagram re£ecting a section over an attached bacterium. B : Small stones of
granite were exposed to £owing groundwater for 3.5 years and in situ hybridized with a Cy-5 labelled probe for the domain Archaea
(ARC-915). A chain of growing archaeal microorganisms is displayed from a top (0³ relative to the light path in the microscope) and a
side view (90³ relative to the light path in the microscope) using the image processing program. The scanned depth was 21.6 Wm. The
depth resolution is about three times less than the side resolution which gives the microorganisms a three times too thick appearance in
the side view. Considering this artefact, it can be concluded that the observed signal is emitted from a threadlike structure with the size
of typical prokaryotic cells growing in chains, presumably a methanogen (see text for details).
K. Pedersen / FEMS Microbiology Reviews 20 (1997) 399^414
409
K. Pedersen / FEMS Microbiology Reviews 20 (1997) 399^414
410
acid probes [20] for the possible in situ detection of
ë spoë groundwaters, inferred by the
methanogens in A
presence of methane (Table 2) with a biogenic signature [19]. A di¤cult problem using £uorescent microscopy on fracture surfaces from granite is the intensive background auto£uorescence from various
accessory fracture coating minerals and precipitates
at short wavelengths typical for DAPI, FITC, acridine orange and rhodamine £uorochromes. We have
solved this problem by using a £uorochrome for the
infrared part of the spectrum, Cy-5, with an excitation maximum at 647 nm and emission above
660 nm. The background £uorescence from various
fracture surfaces is very low with this stain (Fig. 4A).
Groundwater from the Bockholmen site (Fig. 1) carried biogenic methane [19]. One of the boreholes,
KR0013 (68 m), was connected to a 50 l canister
¢lled with 0.5^1 cm large crushed granite that would
act as a substrate for attachment of microorganisms
in the groundwater slowly passing at £ow rates below 0.1 1033 m s31 . In situ hybridization with
a nucleic acid probe for the domain Archaea on
the granitic surfaces after 3.5 years of exposure revealed a positive signal (Fig. 4B). Most likely,
Fig. 4B shows attached methanogens growing in
a chain, as other members of the domain Archaea
generally prefer more extreme pH, salinity or temë spoë
perature conditions than prevailing in the A
granite.
U
6. Biogeochemical processes
6.1. Iron reducing bacteria
Iron reducing bacteria were discovered to be of
major biogeochemical importance in granitic rock
ë spoë
during a block scale redox experiment at the A
HRL. The unexpected redox stability of the studied
system could only be explained by the mobilization
of solid phase ferric iron oxy-hydroxides to liquid
phase ferrous iron by iron reducing bacteria with
organic carbon as electron donor [14,19,35]. We
have isolated several di¡erent bacteria from this habitat able to reduce ferric iron to ferrous iron, including Shewanella putrefaciens [15]. The 16S rRNA gene
sequences show that several of the dominating species sampled from the Bockholmen fracture zone
(Fig. 1) have a 95% or more identity with known
IRB like Pseudomonas medosina [7]. Our results imply that much of the ferrous iron in anoxic groundwater (Table 1) may be a product of microbial iron
reduction and not only due to pure inorganic redox
reactions.
6.2. Sulfate reducing bacteria
Sulfate reducing bacteria frequently appear in the
ë spoë HRL environments at depths greater than apA
proximately 100 m; isolates as well as 16S rRNA
genes related to sulfate reducing bacteria have been
found [15]. Sul¢de production is of particular interest for the disposal of spent nuclear fuel in copper
canisters because sul¢de is the only substance present
in deep groundwater that will cause corrosion of
copper. Oxygen, another copper corrodant, is not
present in deep groundwater and sulfate will not react with copper unless microbes reduce it to sul¢de.
Therefore, evidence and indications of sulfate reduction based on geological, hydrogeological, groundwater, isotope and microbial data in and around
ë spoë HRL tunnel were evaluated by a multidisthe A
ciplinary research group [36] and the most important
conclusions are given below.
Geological data were evaluated to ¢nd the amount
of sul¢de which could be calculated to result from
the sulfate reduction. The conclusion is that the
amount of pyrite normally occurring in the fracture
coatings could explain the amount produced. However, there are other processes in the geological time
span which have also produced pyrite. Therefore, the
existence of pyrite is not a conclusive evidence of
sulfate reduction.
The hydrogeological conditions were evaluated in
order to describe possible transport phenomena related to the sulfate reduction. The questions to be
answered were: Can sulfate reduction take place in
the sea bottom sediments and the resulting sul¢de be
transported with groundwater to the tunnel? Could
the groundwater £ow conditions in the tunnel either
increase or decrease the e¡ect of biological sulfate
reduction? The answer to the ¢rst question is yes,
the process can occur in the sea bed sediments and
the e¡ect on hydrochemistry can be observed in the
water in£ow in the tunnel. Hydrogeological calculations imply a transport time of approximately 100^
K. Pedersen / FEMS Microbiology Reviews 20 (1997) 399^414
400 days for the water passing through the sediments
to reach the tunnel in a proportion of 25%. The
answer to the second question is that the relatively
simple groundwater £ow conditions around the tunnel would not a¡ect the biological process directly.
However, if the sulfate reduction had been an
ancient process, then the e¡ects would soon
be washed out, which has not been the case. In addition, the existence of high bicarbonate and
low sulfate concentrations in the probing holes on
the very ¢rst sampling occasion after the tunnel
was excavated strongly imply that the process is ongoing.
The groundwater chemistry was evaluated by multivariate mixing and mass balance calculations. The
calculations demonstrated that an understanding of
the £uxes of compounds, rather than measurements
of concentrations only, is necessary for modelling
sulfate consumption and bicarbonate production by
SRB. These calculations de¢ned the speci¢c conditions where the process could be ongoing. The results show that a salinity range of 4000^6000 mg l31
of chloride is the optimal one. Sulfate reduction
seems to occur in anaerobic brackish groundwater
with access to dissolved sulfate and organic carbon
or hydrogen. These conditions are mainly found in
the sea bed sediments, in the tunnel section under the
Baltic Sea and in some deep groundwaters, such as
those in the SELECT tunnel section.
Isotope data were expected to give a de¢nite answer to where the sulfate reduction takes place, since
the bacterial processes always result in an enrichment
of the lighter isotopes. Concerning both the N-13 C
and the N-34 S isotopes the results generally point towards the existence of bacterial sulfate reduction.
However, there are several processes in the geological evolution which could have given the same isotopic signatures as well. Therefore, the isotopic data
provide indications of biological sulfate reduction
but no evidence.
Microbiological data were collected in boreholes
where the hydrochemistry indicated an ongoing or
previously ongoing sulfate reduction. The results
show that sulfate reducing bacteria are present,
sometimes in large quantities (Table 3), and that
they can be correlated to a groundwater composition
with high bicarbonate and low sulfate concentrations.
411
7. Hydrogen-dependent microorganisms
7.1. Hydrogen and methane in deep groundwater
Hydrogen is expected to act as an inert gas in most
geochemical reactions and it is therefore usually
overlooked and not analyzed for. Some data on hydrogen in hard rock were recently published [37,38].
Values of 2.2^1574 WM hydrogen in groundwater
from Canadian shield and Fennoscandian shield
rocks was found. Most granitic rocks shows low
but signi¢cant radioactivity which can generate hydrogen by radiolysis of water. Anaerobic mineral
reactions (e.g. anaerobic corrosion of iron) will also
create hydrogen [6]. Finally, deep mantle gases contain hydrogen. Methane occurs frequently in subterranean environments all over the globe and the stable isotope pro¢le commonly indicates a biogenic
origin of the methane. Values of 1.3^18 576 WM
methane in groundwater from Canadian shield and
Fennoscandian shield rocks [37,38] and 1^181 WM
methane in Swedish groundwater have been published previously (Table 3) [8,9,13]. Recent data indicate up to 720 WM methane down to 440 m depth
ë spoë HRL [45]. More support for an ongoing
at A
methane generating process in deep Swedish granite
is provided by the up£ow of gas, mainly methane,
from fracture zones below sea bottom sediments [39^
41]. Pockmarks in Baltic sea sediments have been
observed, indicating gas eruption from fracture systems in the underlying granite.
7.2. Acetogenic bacteria in deep granitic groundwater
Acetogenic bacteria have the capability of reacting
hydrogen with carbon dioxide to acetate, thereby
producing ATP and reducing power for metabolism.
ë spoë tunOne acetogen has been isolated from the A
nel groundwater (Aspo-4). It is a Gram-positive
strictly anaerobic Eubacterium-like species (see Pedersen et al. [15] for details). We have recently found
16S rRNA sequences identical to Aspo-4 also in
ë spoë HRL
groundwater at the SELECT site in the A
tunnel. Pilot experiments with hydrogen addition to
such groundwater resulted in rapid acetate production. The acetate produced can be used by acetoclastic methanogens, iron and sulfate reducing bacteria
and other heterotrophic microorganisms, thereby
K. Pedersen / FEMS Microbiology Reviews 20 (1997) 399^414
412
distribution, numbers and physiological diversity of
the found methanogens are governed by the carbon
dioxide concentration, salinity and organic carbon
content. Oligotrophic, methylotrophic and acetoclastic methanogens dominate in shallow rock (0^190 m)
with relatively high organic carbon content (7.1^
18 mg l31 ), while autotrophic methanogens prevail
in deep boreholes (190^440 m) with a lower (0.9^
4.0 mg l31 ) content of organic carbon.
8. Conclusions ^ The deep hydrogen driven biosphere
Fig. 5. The deep hydrogen driven biosphere hypothesis, illustrated by its carbon cycle. At relevant temperature and water
availability conditions, subterranean microorganisms are theoretically capable of performing a life cycle that is independent of
sun driven ecosystems. Hydrogen and carbon dioxide from the
deep crust of the earth or from sedimentary deposits of organic
carbon can be used as energy and carbon sources. Phosphorus is
available in minerals like apatite and nitrogen for proteins, nucleic acids etc. can be obtained via nitrogen ¢xation ; this gas
predominates in most groundwaters (Table 2).
constituting a transformation route of inorganic carbon to organic carbon with hydrogen as the reductant (Fig. 5).
7.3. Methanogens in deep granitic groundwater
Our results on the presence, diversity and activity
ë spo
of methanogens in 19 di¡erent boreholes at A
ë
HRL (10^440 m depth) are being prepared for publication. Brie£y, the following was found [45]. Pure
cultures of autotrophic, rod like methanogens have
been isolated and 16S rRNA sequencing indicates
them to be related to the genus Methanobacterium
[27]. Viable cell counts (MPN) varied from 10 to
4.3 105 methane producing archaea ml31 . Direct
counts of auto£uorescent cells (£uorescence of the
archaea speci¢c coenzyme F420 ) varied from
1.4 102 to 7.4 105 cells ml31 . Comparisons of total numbers of acridine orange stained cells indicated
that methanogens can constitute up to 60^80% of the
cell population inhabiting the investigated granitic
rock system. The preliminary indication is that the
U
U
U
We have been working on the deep subterranean
biosphere (down to 1240 m) for almost 10 years now
and the list of references shows our progress.
Throughout our work, numerous results have indicated the presence of autotrophic microorganisms
utilizing hydrogen as a source of energy in the
deep environment [12]. We indicated the possibility
of a hydrogen driven biosphere in deep granite in
1992 [8,46], but solid evidence was lacking. Recent
ë spoë HRL tunnel now
results obtained by us in the A
show that autotrophic methanogens, acetogenic bacteria and acetoclastic methanogens are all present
and active in the investigated groundwaters.
Fig. 5 schematically depicts possible routes of carbon and energy in a subterranean hydrogen driven
biosphere. Our present research task is collecting evidence for this model, and we concentrate on archaean organisms and homoacetogenic bacteria.
The presence and activity of iron and sulfate reducing bacteria are well documented [7,14,36] as described above, and have been included in the subterranean biosphere model earlier [5].
Until recently, it has been a general concept that
all life on earth depends on the sun via photosynthesis, including most of the geothermal life forms
found in deep sea trenches as they use oxygen for the
oxidation of reduced inorganic compounds (almost
all oxygen on earth is produced via photosynthesis).
Here, it is suggested that a deep subterranean granitic biosphere exists, driven by the energy available in
hydrogen formed through radiolysis, mineral reactions or by volcanic activity. Knowledge on this biosphere is just beginning to emerge and it will expand
the spatial borders of life from a thin layer on the
surface of the planet Earth and in the seas to a
K. Pedersen / FEMS Microbiology Reviews 20 (1997) 399^414
several kilometers thick biosphere reaching deep below the ground surface and the sea £oor. If this
hypothesis is true, life may have been present and
active deep down in Earth for a very long time,
413
[6] Stevens, T.O. and McKinley, J.P. (1995) Lithoautotrophic
microbial ecosystem in deep basalt aquifers. Science 270,
450^453.
[7] Pedersen, K. and Karlsson, F. (1995) Investigations of subterranean bacteria ^ Their importance for performance assess-
and it cannot be excluded that the place for the
ment of radioactive waste disposal. SKB Technical Report 95-
origin of life was a deep subterranean igneous rock
10. Swedish Nuclear Fuel and Waste Management Co., Stock-
environment (probably hot with a high pressure)
rather than a surface environment. Some of the spe-
holm.
[8] Pedersen, K. (1993) Bacterial processes in nuclear waste disposal. Microbiol. Eur. 1, 18^23.
cies closest to the root of the phylogenetic 16S rRNA
[9] Pedersen, K. and Ekendahl, S. (1992) Incorporation of CO2
tree, as known today, are obligately hydrogen utiliz-
and introduced organic compounds by bacterial populations
ing thermophiles,
in groundwater from the deep crystalline bedrock of the Stripa
pyrus
chaea
belonging
Aquifex pyrophilus and Methanoto the domains Bacteria and Ar-
respectively, supporting the idea of a deep
hot origin of life. A rather spectacular conclusion
is that life on other planets should probably not be
searched for only on the surface but rather deep
down in the subsurface.
mine. J. Gen. Microbiol. 138, 369^376.
î hl, F. and Pedersen, K. (1994)
[10] Ekendahl, S., Arlinger, J., Sta
Characterization of attached bacterial populations in deep
granitic groundwater from the Stripa research mine with
16S-rRNA gene sequencing technique and scanning electron
microscopy. Microbiology 140, 1575^1583.
[11] Ekendahl, S. and Pedersen, K. (1994) Carbon transformations
by attached bacterial populations in granitic groundwater
from deep crystalline bed-rock of the Stripa research mine.
Acknowledgments
The author wishes to thank Johanna Arlinger,
Susanne Ekendahl, Lotta Hallbeck, Louise Holmquist, Nadi Jahromi, Fred Karlsson, Svetlana Kotelnikova and Marcus Laaksoharju, for their contribuë spo
tions and also numerous colleagues at the A
ë
Hard Rock Laboratory and at the Swedish Nuclear
Fuel and Waste Management Co., Stockholm, Sweden, who made our research possible. This work is
supported by the Swedish Natural Science Research
Council, The Swedish Nuclear Fuel and Waste Management Co., and Knut and Alice Wallenbergs
Foundation for Scienti¢c Research.
Microbiology 140, 1565^1573.
[12] Pedersen, K. and Ekendahl, S. (1990) Distribution and activity of bacteria in deep granitic ground waters of southeastern
Sweden. Microb. Ecol. 20, 37^52.
[13] Pedersen, K. and Ekendahl, S. (1992) Assimilation of CO2
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in ground water from southeastern Sweden deep crystalline
bedrock. Microb. Ecol. 22, 1^14.
[14] Banwart, S., Tullborg, E.-L., Pedersen, K., Gustafsson, E.,
Laaksoharju, M., Nilsson, A.-C., Wallin, B. and Wikberg,
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ë spo
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ë spo
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ë
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19, 249^262.
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