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ARTICLE
Timing and origin of natural gas accumulation in
the Siljan impact structure, Sweden
Henrik Drake 1*, Nick M.W. Roberts 2, Christine Heim 3, Martin J. Whitehouse 4, Sandra Siljeström5,
Ellen Kooijman4, Curt Broman6, Magnus Ivarsson4,7 & Mats E. Åström1
Fractured rocks of impact craters may be suitable hosts for deep microbial communities on
Earth and potentially other terrestrial planets, yet direct evidence remains elusive. Here, we
present a study of the largest crater of Europe, the Devonian Siljan structure, showing that
impact structures can be important unexplored hosts for long-term deep microbial activity.
Secondary carbonate minerals dated to 80 ± 5 to 22 ± 3 million years, and thus postdating the
impact by more than 300 million years, have isotopic signatures revealing both microbial
methanogenesis and anaerobic oxidation of methane in the bedrock. Hydrocarbons mobilized
from matured shale source rocks were utilized by subsurface microorganisms, leading to
accumulation of microbial methane mixed with a thermogenic and possibly a minor abiotic
gas fraction beneath a sedimentary cap rock at the crater rim. These new insights into crater
hosted gas accumulation and microbial activity have implications for understanding the
astrobiological consequences of impacts.
https://doi.org/10.1038/s41467-019-12728-y OPEN
1Linnæus University, Department of Biology and Environmental Science, 39182 Kalmar, Sweden. 2Geochronology and Tracers Facility, British Geological
Survey, Nottingham NG12 5GG, UK. 3Department of Geobiology, Geoscience Centre Göttingen of the Georg-August University, Goldschmidtstr. 3, 37077
Göttingen, Germany. 4Swedish Museum of Natural History, P.O. Box 50 007, 10405 Stockholm, Sweden. 5Bioscience and Materials/Chemistry and
Materials, RISE Research Institutes of Sweden, Box 5607, 114 86 Stockholm, Sweden. 6Department of Geological Sciences, Stockholm University, 106 91
Stockholm, Sweden. 7Department of Biology, University of Southern Denmark, Campusvej 55, 5230 Odense, Denmark. *email: henrik.drake@lnu.se
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Impact craters and associated impact-generated hydrothermal
systems may be favorable for microbial colonization on Earth
and potentially other planets1,2. Extensive fracturing at depth
caused by impacts provides pore space, and heat generated by the
impact drives hydrothermal convection, favorable for deep
ecosystems3,4. Although very few studies of the deep biosphere in
impact structures exist, a small number of reports of fossil- and
geochemical signatures support post-impact colonization of
impact hydrothermal systems5,6. However, direct temporal con-
straints of mineralized remains of microorganisms in rock frac-
tures are generally lacking, and the link between the impact and
subsequent colonization thus remains elusive. Confirmation of
impact craters as favorable environments for deep biosphere
communities would substantially enhance our present under-
standing on deep energy cycling of these systems and involve
considerable astrobiological implications3,7. Investigation of
methanogens and methanotrophs are of special interest since
methane emissions, both seasonal and as isolated spikes, have
recently been detected at Martian craters8.
In the largest impact structure in Europe, the Late Devonian
(380.9 ± 4.6 Ma9) Siljan crater in Sweden, the past and present
deep biosphere communities remain unexplored, but the struc-
ture has been thoroughly studied for potential methane accu-
mulation. This strong greenhouse gas can form via three main
mechanisms in the deep subsurface: abiotic, that is, during
inorganic reactions of compounds, e.g. H
2
and CO
2
; thermogenic,
that is, by organic matter breakdown at high temperatures; and
microbial activity10,11, which has been largely overlooked at Siljan
and other impact structures.
In the late 1970s and 1980s, astrophysicist Thomas Gold put
forward controversial theories of mantle-derived methane
ascending through fractures to shallow crustal levels where it
would accumulate and form higher hydrocarbons and
petroleum12,13. Gold proposed that significant amounts of
methane of mantle origin had ascended the impact-deformed
basement at Siljan14, and accumulated beneath a cap rock of
carbonate-sealed fractures in the upper crust. Accordingly, from
the late 1980s to early 1990s, deep exploratory wells were drilled
in the central plateau of exhumed Paleoproterozoic granite15, but
no economic gas quantities could be established and the project
was abandoned. The origin of the hydrocarbons found during the
deep drillings remains disputed, not the least due to potential
contamination from drilling lubricants16. Gold’s theory is now
considered invalid and has been overtaken by newer models on
deep hydrocarbon formation17. Abiotic methane does occur in a
variety of geological settings10, including Precambrian shields, but
the presence of a globally significant abiogenic source of hydro-
carbons has generally been ruled out18. Recent studies of frac-
tured Precambrian crystalline rocks have revealed deep methane
occurrences of various, often complex origin, including microbial,
and abiotic19. High methane concentrations in crystalline rocks
are commonly associated with serpentinized ultramafic and
graphite-bearing rocks10,19, but at Siljan these rock types are not
present and contribution of abiotic methane to the crystalline and
sedimentary rock aquifers is yet to be proven.
In recent years, prospecting for methane has been re-initiated
at Siljan by the prospecting company AB Igrene. This time the
focus is on the fractured crystalline bedrock beneath 200–600 m
thick20 down-faulted Ordovician and Silurian sedimentary rocks
(dominantly limestone but shales are also abundant) in the ring-
like crater depression, where several cored boreholes have been
drilled to 400–700 m depth (Fig. 1)14. Methane accumulations
have been detected during the drilling campaigns, both in the
sedimentary rock (proposed cap rocks) and deep within the
granite fracture system, but no qualified estimate of total gas
volumes has yet been made public.
Occurrences of seep oil associated with the Siljan crater sedi-
mentary successions have, in fact, been known for hundreds of
years, dating back to reports by Linnaeus in 1734in21. Seep oil and
bitumen in limestone have been interpreted to have been gen-
erated from organically rich Upper Ordovician black shale22,23.
The thermal maturity of this organic-rich shale as well as of
Lower Silurian shale has reached the initial stage of oil generation,
and hydrocarbons have migrated from these more mature sedi-
ments into marginally mature sediments24. The overburden and
the time of burial apparently were sufficient to mature the
potential source rocks at Siljan, although it has been speculated
that heat effect of the meteorite impact locally matured the source
rock instantaneously23. Still, the potential input of thermogenic
gas to the deep granite aquifer at Siljan remains elusive.
The potential contribution of microbial methane at Siljan has,
in contrast, largely been overlooked, and, consequently, the deep
microbial communities are yet unexplored, which is the case for
most terrestrial impact structures. Isotopic and biomarker clues to
ancient microbial processes such as methanogenesis and anae-
robic oxidation of methane (AOM) with associated sulfate
reduction can be preserved within minerals formed in response to
these microbial processes. These signatures can remain within the
minerals over considerable geological time25. Relatively light, 12C-
rich, methane is produced during microbial methanogenesis and,
consequently, 13C accumulates in the residual CO
2
26. When these
distinguished carbon pools are subsequently incorporated in
secondary carbonate minerals the isotopic compositions are
preserved such that 13C-enrichment marks methanogenesis27,
and 12C-enrichment AOM28,29. Advances in high spatial reso-
lution U-Pb geochronology make it possible to gain timing
constraints about discrete events of mineral precipitation fol-
lowing methane production and consumption in fractured
rock25,30.
Here, we apply both the carbon and U-Pb isotopic approaches
in combination with analysis of organic compounds and gases to
disclose accumulation of a mixed, but dominantly microbial,
origin of methane of Cretaceous or younger age at Siljan. The
isotopic mineral-gas dataset is the most comprehensive yet
reported from any impact structure and provides new constraints
for the unexplored deep microbial ecosystems of terrestrial
impact craters, particularly regarding deep methane formation
and consumption. Implications include both a broader perspec-
tive regarding natural gas accumulations in the upper crystalline
crust, and the potential and challenges in understanding the
significance of impacts as oases for life on otherwise dead pla-
netary bodies.
Results
Stable carbon isotope composition of calcite. Calcite that occurs
together with sulfides and bitumen in secondary mineral coatings
of open fractures (Figs. 2and 3) shows a large δ13C variability, in
total 73.8‰V-PDB (n=984, Supplementary Data 1). The values
are ranging from significantly 13C-depleted (−52.3‰)to13C-
enriched (+21.5‰). Many of the δ13C excursions occur where
extensive gas accumulations were observed during drilling
(Fig. 4). The δ13C
calcite
range in the sedimentary rock fractures is
−12.5‰to +21.5‰and in granite −52.3‰to +18.9‰.
Strongly positive δ13C
calcite
values occur in 38% of the fractures in
the sedimentary rock and 28% in the granite, and are found up to
212 m above the sediment-granite contact (177 m below ground
surface), as well as up to 214 m below the contact, to maximum
depths of 620 m. The most 13C-depleted calcite (−52.3‰) is from
the granite-sedimentary rock contact (Fig. 4a, b).
Samples with large 13Cdepletion(δ13C<−35‰)or13C-
enrichment (δ13C>+5‰) either show relatively homogeneous
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composition throughout the crystals (Fig. 2o, r), or 13Cenrichment
or depletion at the outermost growth zones (Fig. 2p, q). The latter
feature is accompanied by a significant increase in 87Sr/86Sr values
in the outermost growth zone compared to the inner part (Fig. 2q),
in contrast to the relatively homogeneous 87Sr/86Sr values of calcite
with homogeneous δ13C values (Fig. 2r). Calcite in limestone
fractures with abundant solid bitumen has highly positive δ13C
values.
Stable sulfur isotope composition of pyrite. The δ34S
pyrite
values
in fractured granite span 119.8‰, from −41.9‰to +78.0‰
(Supplementary Data 2, n=443). In sedimentary rock fractures
the values are from −5.8‰to +41.8‰. The δ34S
pyrite
value
distribution within and between different crystals in the same
fractures shows large variability. Notable examples include rela-
tively homogeneous light δ34S
pyrite
values of −40 ± 1‰(Supple-
mentary Fig. 1b), increasing δ34S
pyrite
values from core to rim
(Supplementary Fig. 1c), and small variation within individual
crystals but substantial variation (93.7‰) between different
crystals (Supplementary Fig. 1d).
Fluid inclusions. Only one of ten calcite samples examined
contained fluid inclusions (Supplementary Data 3). These inclu-
sions are of one and two-phase type. The latter have homo-
genization temperatures of 40–55 °C and ice melting
temperatures equivalent to salinities of 1.6–2.7 mass % NaCl
(n=6). The general lack of fluid inclusions and the nature of the
few detected inclusions indicate low-temperature origin.
U-Pb geochronology. Seven calcite samples gave U-Pb age solu-
tions from the high spatial resolution analyses. 13C-enriched calcite
from a limestone fracture at 212 m depth gave a single event age of
22.2 ± 2.5 Ma (Fig. 5b) whereas the 13C-enriched calcite at 170 m is
more complex, with two isochrons; at 80 ± 5 Ma and 39 ± 3 Ma
(Fig. 5a). In the granitic basement, ages of 39.2 ± 1.4 Ma and 65 ±
10 Ma were obtained when targeting the 13C-rich outermost calcite
growth zones at 537 and 442 m depth, respectively (Fig. 5c, d).
13C-depleted calcite at the sediment-granite contact gave a 37.7 ±
1.9 Ma age but also an uncertain population at 464 ± 60 Ma
(n
spots
=3, Fig. 5e). Calcite without any significant excursions in
δ13C
calcite
values (−3.9 to +0.8‰) from two granite fracture
samples yielded 506 ± 25 and 576 ± 64 Ma ages (Supplementary
Fig. 2, full data and analytical details in Supplementary Data 4–6).
Organic remains in the mineral coatings. The calcite coatings
analyzed for preserved organic compounds using gas-
chromatography mass spectrometry (GC-MS, n=6) repre-
sented 13C-rich calcite in limestone (VM2:212) and granite
(CC1:539 and 608, VM1:442), as well as 13C-depleted calcite from
the granite-sedimentary rock contact (VM1:251). Although
overall low in organic content, the sample from limestone showed
a clear unimodal distribution of n-alkanes ranging from n-C
17
to
n-C
42
with a maximum at n-C
23
(Fig. 6a). The hydrocarbon range
ab
15°17′12′′E
14°25′47′′E
60°50′33′′N
N
NRoads
8 km
500 km
Siljan
Lake
Skattungen
Lake
Ore
Lake
Orsa
Lake Siljan
Solberga1
Solberga
Quarry
Rättvik
town
Stumsnäs1
Mora town
CC1
VM1
VM2
01–10C
01–11C
Orsa town
Precambrian crystalline rocks
Late Silurian Orsa sandstone
Drill sites (with borehole name)
Ordovician and Silurian sediments
(Tremadocian to Wenlock)
SWEDEN
61°14′10′′N
Fig. 1 Maps of the Siljan impact structure and study locations. aMap of Sweden with the Siljan area indicated. bGeological map of the Siljan impact
structure with locations of the cored boreholes and the quarry sampled for mineral coatings indicated, along with the sedimentary units in the crater
depression, towns, lakes (white) and roads (black lines). Gas compositions exist from boreholes VM2 and VM5 (located adjacent to VM2). Modified
from ref. 68
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at the sediment-granite contact was n-C
17
to n-C
36
, with a similar
unimodal distribution as in the limestone, but with a peak
maximum at C
25
. Two of the granite samples (VM1:442 and
CC1:539) had the same hydrocarbon distribution as in the rock
contact, although the general abundance was lower and with
distinct peaks of n-C
18
,n-C
20
and n-C
22
and n-C
28
. The deepest
sample, CC1:608, showed hydrocarbons ranging from n-C
18
to
n-C
32
with a maximum at C
20
and low abundances of n-C
26
to
n-C
32
. Pristane (Pr) and Phytane (Ph) were present in the lime-
stone and the two granite CC1 samples (Supplementary Data 7).
The Pr/Ph, Pr/n-C
17
and Ph/n-C
18
ratios were relatively low (<1).
In CC1:539, small amounts of C
27
to C
35
hopanoids, partly S and
R isomers were detected (Fig. 6c). Fatty alcohols n-C
14
-OH; n-
C
16
-OH, and n-C
18
-OH were detected in all samples, in varying
intensities (Fig. 6b). Mono ether lipid (1-o-n-hexadecylglycerol)
was detected in VM1:251. The samples contained fatty acids (FA)
0
5
10
15
20
25
1234
0.714
0.716
0.718
0.72
–50
–48
–46
–44
–42
–40
–38
–36
–34
–32
–30
1234567
δ13C
–10
–5
0
5
10
123456
2
1
3
45
6
123
δ13C (‰ V-PDB) δ13C (‰ V-PDB)
δ13C (‰ V-PDB) δ13C (‰ V-PDB)
Spot number in transect
k
2
3
1
5
6
4
l
Calcite
Pyrite
Pyrite
d
Calcite
Qz
g
b
7
a
Pyrite
Calcite
ef
Calcite
1
23
1
2
3
4
k
j
j
Calcite
Calcite
c
2
3
1
4
5
6
0.72
0.722
0.724
0.726
0.728
0.73
0.732
–10
–5
0
5
10
15
20
123456
87Sr/86Sr
87Sr/86Sr
87Sr/86Sr
87Sr/86Sr
h
i
m
n
o
p
q
r
δ13C
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ranging from n-C
12
to n-C
24
, with a clear even over odd pre-
dominance and high abundances of n-C
16
and n-C
18
FA (Fig. 6b).
Odd and branched FAn-C
15
,ai-C
15
, 12Me-C
16
,ai-C
17
, and n-C
17
and 12OH-C
18
, as well as mono-unsaturated fatty acids C
18:1
and
n-C
16:1
were also detected.
Time of flight secondary ion mass spectrometry (ToF-SIMS)
performed to putative organic material in granite sample
CC1:537 showed a negative spectra with peaks at m/z 255.2,
283.2, and 281.2 assigned to fatty acids C
16:0
,C
18:0
, and C
18:1
31,
consistent with the GC-MS data. The positive spectra showed
peaks that can be assigned to polycyclic aromatic hydrocarbon
(PAHs, Supplementary Figs. 3 and 4).
Composition of modern gas. The gas encountered during bore-
hole drilling in the sedimentary rock (VM2, gas and water samples)
and in a borehole section covering sedimentary rock down below
the granite contact (VM5, gas samples), is dominated by methane
(mainly >90%, whereas CO
2
is at 3–14% in the gas samples, with
even higher CO
2
in the water samples). The ratio of methane to
higher hydrocarbons, C
1
/(C
2
+C
3
), is 125–200 (Supplementary
Data 8) and there are notable relative concentration patterns: C
2
>
C
3
,iso-C
4
>n-C
4
,i-C
5
>n-C
5
,andneo-C
5
>i-C
5
.Themethanehas
δ13Cvaluesof−64 ± 2‰which are lighter than the δ13C
C2
(−28 ±
2‰)andδ13C
C3
values (−7‰). The δ2H
CH4
value in the single
sample analyzed for this ratio is −240‰SMOW for the VM5
borehole gas and δ13C
CO2
values are c. +5–8‰.
Discussion
The highly variable δ13C
calcite
values between different fractures
and within single crystals point to spatiotemporal variation of the
processes that lead to calcite precipitation. We focus our discus-
sion on processes producing the youngest calcites, which feature
large δ13C
calcite
excursions. The older type predates the impact
and dates back to 600–400 Ma (Supplementary Fig. 2) and shows
no δ13C signatures diagnostic for methane cycling. In order to
link the mineral data to the present gas in the system, the dis-
cussion starts with interpretations of the gas compositions that
exist from the new boreholes and from previous prospecting.
Interpretation of the origin of hydrocarbon gases is typically
based on diagnostic geochemical signatures, normally by using
discrimination diagrams (Fig. 7, based on a global compilation32)
and a holistic approach including the geological context. The
most widely used discrimination diagram is the ratio of methane
to higher hydrocarbons, C
1
/(C
2
+C
3
), versus δ13C
CH4
(Fig. 7a).
This differentiates microbial gas which usually has high C
1
/(C
2
+
C
3
) (>1000)26 from the typically lower ratios of thermogenic
methane (<50)33,34. However, abiotic gas may also show high C
1
/
(C
2
+C
3
)35,36 and cannot be excluded based on this ratio. For
δ13C
CH4
, there is typically a difference between methane sources,
ranging from the substantially 13C-depleted microbial, through
moderately 13C-depleted thermogenic to isotopically heavier
abiotic methane26. Microbial methanogenesis can be divided into
Fig. 2 Appearance and paragenesis of calcite and transects of δ13C and 87Sr/86Sr values. a–dDrill core photographs of fractured core sections with notable
gas observations (according to drilling logs), SEM images of secondary minerals on the fracture surfaces (back-scatter electron [BSE] mode, (e–i), polished
crystal cross-sections with transects of SIMS analyses indicated (j–n) and δ13C(o–r) and 87Sr/86Sr values (q,r) corresponding to the spot locations in j–n.
Details: aSample VM2:170 m, limestone with open fractures. eScalenohedral calcite crystals intergrown with cubic pyrite VM2:170 m. fScalenohedral
calcite crystals from a similar limestone fracture at a slightly greater depth, VM2:212m. j,kpolished crystals from eand f, respectively (corresponding δ13C
values in o, all 13C-enriched). bSample CC1:537, with euhedral calcite on a fracture in porphyritic crystalline rock coated by euhedral quartz (c). The calcite
shows growth zonation (l) and is 13C-enriched in the outer parts (p). cSample VM1:442 m, open fracture in heavily fractured crystalline rock section
coated by aggregates of euhedral calcite (h). The calcite shows growth zonation (m) and is heavily enriched in 13C and 87Sr in the rim compared to the
older growth zones (q). d) Sample VM1:255 m, fractured section of crystalline rock with anhedral calcite coating with pyrite (i). The calcite shows growth
zonation (n) but relatively homogeneous 13C-depleted δ13C values and 87Sr/86Sr (r). Errors (2σ) are within the size of the symbols if not visible. Length of
scale bars: (a–d) 4 cm, (e,k) 400 µm, (f,h,i,j,n) 500 µm, (g,l,m) 300 µm
Calcite
Asph
Asph
Pyrite
Calcite
Asph
a
b
fg
j
e
Calcite
Asph/oil
cd
Fig. 3 Appearance and paragenesis of solid bitumen and seep oil. (a–d)
from sedimentary rock fractures (e–g) from crystalline rock fractures.
Drill core photographs are shown in (a,c,e) and SEM images of secondary
minerals on the fracture surfaces (BSE mode) are shown in (b,d,f,g).
aWhite limestone (VM2:328 m) with abundant solid bitumen
(asphaltite =“Asph”) and oil seep. bAbundant scalenohedral calcite
(13C-rich) crystals on the fracture in (a), and small patches of bitumen
(“Asph”). cFractured section of limestone (01–10C:326 m) with abundant
occurrences of solid bitumen and oil seep. dEuhedral calcite (13C-rich)
crystals on the fracture in (a) with abundant bitumen and seep oil smeared
on the surface. eDrill core sample VM1:255 m (fractured crystalline rock).
fEuhedral pyrite and bitumen on the fracture surface (e). gBitumen
(“Asph”) and calcite (slickensided and partly euhedral crystals) in an
adjacent fracture, VM1:251 m. Length of scale bars: (a,c,e) 4 cm, (b,f,g)
500 µm, (d)1mm
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the carbonate reduction pathway and acetate (methyle-type)
fermentation37, of which the former involves a larger kinetic
carbon isotope effect26. At Siljan, a dominantly microbial gas
fraction is suggested by the light δ13C values of the methane
(−64 ± 2‰, Fig. 7a). However, the C
1
/(C
2
+C
3
) values are
slightly lower than expected from a pure microbial gas and
therefore point to a mixed origin, as indicated by the position at
the border between the microbial carbonate reduction and early
mature thermogenic fields (Fig. 7a). Regarding the higher
hydrocarbons, it has been demonstrated that microbial ethano-
and propanogenesis occur in deeply buried marine sediments38,
and the former also near gas wells in western Canada39. Presence
of ethane and propane is thus not a definite marker for ther-
mogenic gas. However, in a microbial gas, the presence of C
4+
gases (detectable n-C
4
,i-C
4
,i-C
5
, Supplementary Data 8) is not
expected33,34, and the δ13C
C2
values are typically not as heavy as
those measured (−28 ± 2‰)40, in particular in comparison to the
light δ13C
CH4
values (−64 ± 2‰), which indicate thermogenic
contribution. These relatively heavy δ13C
C2
values and even
heavier δ13C
C3
values speak against a significant contribution
from abiotic gas, which generally features decreasing δ13C values
with higher carbon number of the homologues41. At other
igneous rock sites in South Africa, Canada, and Scandinavia,
abiotic methane shows lighter δ13C values (−50‰10,41) than the
typically assigned values of abiotic methane (i.e. >−20‰42),
although not as low as the methane at Siljan.
The isotopic hydrogen signature (δ2H
CH4
)of−240‰
SMOW for the VM5 borehole gas is, when plotted against
δ13C
CH4
(Fig. 7b) also in the microbial carbonate reduction
field32, however, close to early mature thermogenic gas and
0
50
100
150
200
250
300
Depth (m)
Depth (m)
350
400
450
500
550
600
650
0
50
100
150
200
250
300
350
400
450
500
550
600
650
0
50
100
150
200
250
300
350
400
450
500
550
600
650
0
50
100
150
200
250
300
350
400
450
500
550
600
650
VM-2
01–10C
01–11C
a
Sedimentary rock
Crystalline rock
Gas
Oil
Solberga-1
d
Crystalline rock
Sedimentary rock
b
Crystalline rock
Sedimentary rock
–55
–50
–45
–40
–35
–30
–25
–20
–15
–10
–5
0
5
10
15
20
25
–55
–50
–45
–40
–35
–30
–25
–20
–15
–10
–5
0
5
10
15
20
25
–55
–50
–45
–40
–35
–30
–25
–20
–15
–10
–5
0
5
10
15
20
25
–55
–50
–45
–40
–35
–30
–25
–20
–15
–10
–5
0
5
10
15
20
25
CC-1
c
Sedimentary rock
Crystalline rock
δ13Ccalcite (‰ V-PDB)
δ13Ccalcite (‰ V-PDB)
VM-1VM-2
Fig. 4 δ13C
calcite
vs depth. aClosely spaced boreholes VM2, 01–10C and 01–11C (samples from the latter two have been depth-normalized to the
sedimentary-crystalline rock interface in VM-2 in order to plot them in the same graph), (b) VM1, (c) CC1, and (d) Solberga-1, with Solberga quarry
samples collected at close to ground surface, ~400 m south of Solberga-1 drill site. Each data point represents one SIMS analysis. Fractures with gas
occurrences observed during drilling are marked with “x”(including a seep oil observation from borehole 01–10C/VM3, marked “−“) on the right-hand side
of the graphs and the depth of the sedimentary-crystalline bedrock interface is also indicated. Errors (2σ) are within the size of the symbols. All plots have
the same range on the axes for comparison
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fermentation type microbial gas. Additionally, the Siljan gas
samples are overlapping with δ2H
CH4
ranges of abiotic sources
at other sites10, meaning that abiotic contribution cannot be
ruledoutbasedontheδ2H
CH4
composition. Overall, the
position of the gas samples at borders or within multiple
empirically defined zones on the discrimination plots (Fig. 7a,
b) shows that these plots alone are not diagnostic for any single
processand/orgasorigin.
The heavy δ13C
CO2
(c. +5–8‰, Fig. 7c, Supplementary
Data 8) of the Siljan samples is typical for microbial methano-
genesis through carbonate reduction32 and thus another feature
supporting a dominantly microbial gas origin. These δ13C
CO2
values are characteristic for secondary microbial methane43
formed following microbial utilization of primary thermogenic
hydrocarbons (e.g. petroleum, seep oils and lighter hydro-
carbons), which is supported by other biodegradation signatures.
These signatures include high C
2
to C
3
ratios owing to that ethane
is relatively resistant to biodegradation compared to the C
3+
homologues44. Biodegradation also discriminates against 13C
C3
,
leading to isotopically heavy residual propane44. In the Siljan gas,
the anomalously heavy δ13C
C3
values compared to the δ13C
C2
values, the high C
2
to C
3
ratios that are far from the normal range
for thermogenic gases, as well as other signatures presented in
Supplementary Note 1, thus point to biodegradation, but to an
unknown degree. The removal of the higher hydrocarbons during
biodegradation increases the C
1
/(C
2
-C
3
), which complicates the
estimation of the mixing proportions between microbial and
thermogenic gas.
0.0
0.2
0.4
0.6
0.8
0 40 80 120 160 200
Blue = Chip B+C+D+F
Intercept at 80 ± 5 Ma
(MSWD = 3.0; 1 rejected)
Red = Chip A+E+G
Intercept at 39 ± 3 Ma
(MSWD = 10.8; 1 rejected)
Green = Chip H
No regression
VM2:170
207Pb/206Pb
207Pb/206Pb
238U/206Pb
238U/206Pb
238U/206Pb
207U/206Pb
0.0
0.2
0.4
0.6
0.8
1.0
0 40 80 120 160 200
Intercepts at
39.2 ± 1.4 Ma
MSWD = 2.1
CC1:537
0.0
0.2
0.4
0.6
0.8
0 40 80 120 160 200
Blue = younger
Intercepts at 37.7 ± 1.9 Ma
(MSWD = 0.92)
Red = older
Interceptsat 464 ± 60 Ma
(MSWD = 0.08)
VM1:255
a
b
d
e
0.4
0.5
0.6
0.7
0.8
0.9
1.0
0 20 40 60 80 100 120 140
Intercept at 22.2 ± 2.5 Ma
(MSWD = 1.2; 1 rejected)
VM2:212
0.0
0.2
0.4
0.6
0.8
1.0
0 20406080
Blue = Chip C+D+E+L+M
Intercept at 65 ± 10 Ma
(MSWD = 2.1; 1 rejected)
Red = Chip A+B+H+I+J
No regression
0
VM1:442
c
Fig. 5 U-Pb carbonate dating. aVM2:170, two generations of 13C-rich calcite in limestone fracture, yielding ages 80 ± 5 Ma and 39 ± 3 Ma. bVM2:212 13C-
rich calcite in limestone fracture, yielding 22.2 ± 2.5 Ma. cCC1:537, 13C-rich calcite, in granite fracture, yielding 39.2 ± 1.4 Ma. dVM2:442, 13C-rich calcite
in granite fracture, yielding 65 ± 10 Ma. eVM1:255, 13C-depleted calcite in granite fracture (targeting the growth zones with dark BSE-intensity in Fig. 2n),
yielding 37.7 ± 1.9 Ma, with an older potential event 464 ± 60 Ma (highly uncertain: only three data points). Errors represented by the ellipses are 2σ
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RT: 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
Time (min)
0
20
40
60
80
100
0
20
40
60
80
100
20
40
60
80
100
20
40
60
80
100
20
40
60
80
100
VM2:212
VM1:251
VM1:442
CC1:539
CC1:608
a24
18
18
18
18
c
c
Relative intensity
34
RT:15.16 – 40.66
20 30 40
Time (min)
20
60
100
RT: 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
Time (min)
0
5
10
0
5
10
0
5
10
0
5
10
0
5
10
VM2:212
VM1:251
VM1:442
CC1:539
CC1:608
14:0
16:1 18:1
15:0
ai
i
ai
i
12Me-16:0
12Me-16:0
12OH-18:0
b
ai
12Me-16:0
12OH-18:0
12OH-18:0
Relative intensity
Relative intensity
18:1
18:1
ai
i
i
36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53
Time (min)
0
10
20
30
40
50
60
70
80
90
100 17α,21β(H)-C30
17α,21β(H)-C29 17α,21β(H)-C31 S+R
17α,21β(H)-C32 S+R
17α,21β(H)-C33 S+R
17α,21β(H)-C34 S+R
17α,21β(H)-C35 S
17α(H)-C27
CC-1: 539
c
34
12OH-18:0
ai
ai
ai
i
ai
16:1
16:1
16:1
ai
i
12OH-18:0
c
Relative abundance
20
60
100
20
60
100
20
60
100
20
60
100
Fig. 6 Partial mass chromatograms. Samples are from limestone fracture (VM2:212), sediment-granite contact (VM1:251) and deep granite fractures
(VM1:442, CC1:539, CC1:608), showing (a) the n-alkane distribution pattern (straight chain hydrocarbons, m/z 85, ●), (b) the fatty acid distribution (m/z
69, 74; ■) of all investigated samples and (c) weak hopanoid signals (m/z 191) from the calcite in the granite fracture sample CC1:539
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All the gas data together thus point to a gas that is to a large
extent microbial and to a significant extent thermogenic.
Although abiotic gas contribution cannot be directly identified in
the investigated (dominantly sedimentary) aquifer, it cannot be
ruled out, at least not in the deep granite fracture system, because
none of the borehole samples isolates gas from the crystalline
aquifer alone. Theoretically, abiotic gas contribution from the
granite fractures may thus be masked by gases from the sedi-
mentary rock fractures. Variably depleted δ13C
CH4
signatures
from previous investigations16,45, summarized in Supplementary
Note 2, are in accordance with the mixed gas of interpreted
dominantly microbial and thermogenic origin detected in the
present study (Fig. 7). Hence, although the results presented here
and previously are not generally supportive of abiotic gas, such
gas cannot be fully ruled out, at least not in the deeper, granitic
system.
For the mineralogical record, the significantly 13C-enriched
calcite observed in the fractures in limestone (δ13C
calcite
values as
heavy as +21.5‰, Fig. 4b) and granite (up to +18.9) is evidence
for formation following microbial methanogenesis in situ, owing
to the discrimination that occurs against 13C during methano-
genesis that leaves 13C-rich residual carbon behind25,27,46.
However, the presence of 13C-rich calcite cannot completely rule
out minor abiotic gas fractions. In addition, the FA n-C
12
to n-
C
18
, particularly the odd chain and branched iC
15
, aiC
15
,n-C
15
,
12Me-C
16
, aiC
17
, and 12OH-C
18
as well as the n-alcohols and the
1-o-n-hexadecylglycerol preserved within 13C-rich, methano-
genesis-related, calcite coatings are support for in situ microbial
activity. These preserved FA can be tied to fermentation47, sulfate
reduction by bacteria48 and other microbial processes (Supple-
mentary Note 3), but not specifically to methanogens (archaea),
which do not produce phospholipid fatty acids.
The U-Pb ages suggest that methanogenesis in the sedimentary
and granite aquifers at Siljan led to precipitation of 13C-rich
calcite on several occasions, from 80 ± 5 to 22.2 ± 2.5 Ma (Fig. 5).
The distribution of this calcite marks microbial methanogenesis
in the upper 214 m of the fractured crystalline rock in the crater
structure (to depths of 620 m) and in the overlying sedimentary
rock fractures over a depth span of more than 200 m. The sub-
stantial 13C
calcite
and 13C
CO2
enrichments occurring in the lime-
stone aquifer are noteworthy, because dilution by the C isotope
signature of dissolved inorganic carbon (DIC) derived from
limestone (δ13C: 0 to +2‰49) would be expected. To explain this
feature, we propose local influence from kinetic microbial pro-
cesses on the δ13C
DIC
signature in the Siljan aquifer, in common
with observations from other deep energy-poor fracture
system28,50 (Supplementary Note 4). This phenomenon should be
particularly important in pore space infiltrated by gases, bitumen
or seep oils, as shown by spatial relation of these features to
significantly 13C-rich calcite (Fig. 3). Preserved hydrocarbon n-
alkane pattern of calcite in bitumen-bearing fractures of the
sedimentary rock and at the sediment-granite interface
(VM2:212; VM1:251, Fig. 6) is indication for thermal- and bio-
degradation. It has previously been reported that biomarkers in
bitumen in sedimentary rock fractures link its origin to shales and
that mobilization and degradation of hydrocarbons have occurred
on several events in the fracture systems22 (additional biomarker
support in Supplementary Note 1).
Methanogenesis is commonly associated with sulfate-poor
biodegraded petroleum reservoirs51 and initial steps of anaerobic
utilization of organic matter (fermentation) involve hydrolysis
followed by bacterial acetogenesis that converts volatile fatty acids
into acetic acid, H
2
and CO
2
52. Alternatively, H
2
is produced by
aromatization of compounds present in the seep oil51. Metha-
nogenesis through CO
2
reduction, with H
2
as electron donor, has
been proposed to be the dominant terminal process in petroleum
biodegradation in the subsurface47, and this appears also to be the
case at Siljan based on the widespread and pronounced heavy
δ13C
calcite
and δ13C
CO2
values (Figs. 4and 7c). In sulfate-rich
reservoirs, microbial sulfate reduction (MSR) can be involved in
degradation of hydrocarbons. In the Siljan fractures, pyrite
occasionally occurs together with 13C-enriched calcite. Pyrite
formed by MSR is typically strongly depleted in 34S53. The very
low minimum δ34S
pyrite
values (−40‰V-CDT, Supplementary
Fig. 1) is thus proposed to reflect MSR. However, groundwater in
granite fractures of adjacent boreholes show very low sulfate
concentrations, 4.3–6.6 mg L−145, suggesting a generally low
potential for MSR in that aquifer. Although anaerobic oxidation
of organic matter by MSR can produce CO
2
that can be utilized
by methanogens43, it did probably not result in large quantities of
methane because sulfate reducers outcompete methanogens for
H
2
and other substrates when sulfate concentrations are
10
–1
10
0
10
1
10
2
10
3
10
4
10
5
C
1
/(C
2
+C
3
)
Primary
microbial F
SM LMT
CR
OA
EMT
Abiotic
Thermogenic
δ
13
C
CH4
(‰)
δ
13
C
CH4
(‰)
δ
13
C
CO2
(‰)
δ
13
C
CH4
(‰)
δ
2
H
CH4
(‰)
Primary microbial
Thermogenic
CR
T
T
OA
EMT
Abiotic
FSM
LMT
Thermogenic
Abiotic
LMT
OA
EMT
CR
Primary
microbial
F
SM
–90
–80
–70
–60
–50
–40
–30
–20
–10
0
10
20
–450
–400
–350
–300
–250
–200
–150
–100
–50
0
abc
–90
–80
–70
–60
–50
–40
–30
–20
–10
0
10
–50
–40
–30
–20
–10
0
10
20
30
40
–90
–80
–70
–60
–50
–40
–30
–20
–10
0
10
Fig. 7 Gas composition discrimination diagrams. (a)δ13C
CH4
versus C
1
/(C2 +C
3
). (b)δ2H
CH4
versus δ13C
CH4.
(c)δ13C
CH4
versus δ13C
CO2
(adapted
from32). Position of the gases from boreholes VM2 (diamonds =gas samples, triangle =water samples), VM5 (square) and drinking water well (circle)
are shown. Genetic gas field abbreviations denote: CR CO
2
reduction, F methyl-type fermentation, SM secondary microbial, EMT early mature
thermogenic, OA oil-associated thermogenic, LMT late mature thermogenic gas. Gas data were extracted from the database of AB Igrene (Supplementary
Data 8), and from45 (drinking well at Gulleråsen close to Solberga, δ13C
CH4
:−60.3‰,δ2H
CH4
:−269‰). The sampling site of the gas data in borehole
VM2 corresponds to the uppermost 13C-rich calcites in this borehole, whereas the other gas-sampled borehole (VM5) is just adjacent to other boreholes
sampled for calcite (VM- and 01-boreholes). Errors (2σ) are within the size of the symbols
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elevated54. Instead, fermentation likely dominated initial degra-
dation steps of organics in the system providing H
2
for the
methanogens to perform reduction of CO
2
. Furthermore, the low
salinity in the deep granite aquifer45 is favorable for microbial
methanogenesis55. Secondary methane formation following
microbial utilization of primary thermogenic hydrocarbons
typically involves large 13C-enrichment in carbonate32, as mani-
fested by heavy δ13C
calcite
(Fig. 4) and δ13C
CO2
(Fig. 7c).
Overall, the geological setting with relatively low temperatures
and shallow reservoir with abundant seep oil/bitumen are, toge-
ther with gas signatures and 13C-enriched calcite, in favor of
formation of secondary microbial methane produced following
biodegradation of thermogenic hydrocarbons (gas, seep oil and
bitumen). The primary thermogenic gas remains as a minor
biodegraded mixing fraction, in common with secondary
methane reservoirs elsewhere32,43. In the deeper granite system,
contribution from abiotic gas sources may also have been
involved.
During AOM, a phenomenon where methanotrophs can act in
syntrophic relationship with MSR, carbonate may precipitate and
inherit the significantly 13C-depleted signatures of the methane56.
The light δ13C
calcite
values detected at the sediment-granite con-
tact at Siljan (−52.3‰, Fig. 4) are thus proposed to reflect AOM
(Supplementary Note 5 describes more moderately 13C-depleted
calcite). The U-Pb age of this calcite shows that AOM dates back
at least 39 ± 3 Myr. The δ13C
calcite
values point to utilization of
methane of dominantly microbial origin10,26, because thermo-
genic and abiotic methane are usually heavier57. The δ13C sig-
nature of carbonate originating from oxidized methane is
typically diluted by other relatively 13C-rich dissolved carbon
species prior to incorporation in calcite29. When taking such
dilution into account, it is likely that the source methane was
isotopically light, in line with the δ13C
CH4
composition (−64 ±
2‰) of dominantly microbial origin (with a minor thermogenic
and possibly a minor abiotic component) in boreholes VM2/5.
Furthermore, in the sample with the most 13C-depleted (AOM-
related) calcite, co-genetic pyrite has low minimum δ34S
pyrite
values (−18.7‰, Supplementary Fig. 1) reflecting large 32S
enrichment characteristic for MSR-related sulfide53. This finding,
together with MSR-related58,59 branched fatty acids (ai-C
15:0
,
12Me-C
16:0
,ai-C
17:0
, 12OH-C
18:0,
Fig. 6b) mark coupled AOM-
MSR at the sedimentary-granite rock contact. Another note-
worthy feature is the overall large δ34S
pyrite
spans that mark MSR-
related reservoir effects throughout the fracture system (Supple-
mentary Fig. 5 and Note 5).
Several precipitation events are recorded by the intra-crystal
variability of the C and Sr isotopes, and the U-Pb age groups. Our
interpretation is that these precipitation events were caused by
fracture reactivations, as presented in detail in Supplementary
Note 6. In summary, there are tectonic events in the far-field and
uplift events that temporally coincide with the ages of the
methane related calcite at Siljan. Methane cycling can thus be
related to these fracture reactivation events that are more than
300 million years younger than the impact.
A conceptual model for methane accumulation in the Siljan
impact structure, as outlined in Fig. 8, has its basis in the isotopic
inventory of secondary fracture minerals and gases. The microbial
methanogenic processes date back at least to the Late Cretaceous
(Fig. 5), although there are fractures in the granite that formed
significantly earlier (Supplementary Fig. 2). The gas compositions
corroborate that the gas in the sedimentary reservoir is microbial
with contribution from a biodegraded thermogenic end-member
linked to thermal maturity of black shales in the sedimentary pile,
and perhaps a minor abiotic gas fraction. In the upper part of the
sedimentary successions there is local seepage to the surface, as
shown by methane in drinking water wells45. The isotopic
composition of methane in such a well45 fits with both microbial
and early mature thermogenic origin (Fig. 7b). Bitumen can be
mobilized when thermally affected60. Bitumen and seep oil
migration from the organic-rich shales into other sedimentary
rock units and into the fractured granitic basement have thus
likely been initiated when the sediments were thermally matured,
either as a result of the heat from the impact23 or due to sub-
sidence related to Caledonian foreland basin crustal depression61.
The bitumen and seep oil occurrences (along with migrated
thermogenic gas) provided energy for the indigenous microbial
communities in the deep subsurface, as shown by the spatial
relation to 13C-rich calcite. Deep abiotic gas contribution to the
methane accumulations in the granite fractures cannot be ruled
out. However, the apparently higher (but not yet quantified)
abundance of gas beneath the sedimentary rock in the crater rim
(Fig. 4) than in the central dome16, the input of shale-derived
hydrocarbons to the granite fractures, and the similar 13C-
enrichment of calcite in granite and sedimentary fractures point
to similar formation and accumulation of methane in the granite
fracture network as in the sedimentary rock. The dominantly
Eocene–Miocene ages of the 13C-rich calcite indicate that the
major microbial utilization of the hydrocarbons in the deep
fractures occurred when temperatures were more favorable (<50 °
C) for microbial activity, in line with the uplift and subsidence
history of the south-central Fennoscandian shield62. The
Eocene–Miocene microbial activity is proposed to be linked to
regional re-opening of bitumen-bearing fracture sets. This
enabled circulation of groundwater along flow paths with sub-
strates accessible to the microbes in the form of bitumen/oil
coatings, as well as facilitated circulation of biodegradable ther-
mogenic gas in the deep reservoir. The spatial relation of 13C-
enriched calcite and biodegraded bitumen/seep oil suggests sec-
ondary methane formation following anaerobic degradation of
organic matter. This fermentation process produces H
2
for uti-
lization by methanogens through reduction of CO
2
formed dur-
ing biodegradation or occurring in the aquifer. The kinetic
microbial processes producing methane resulted in large isotopic
fractionations, as observed in the gases and secondary carbonates.
Taken together, there are numerous lines of evidence in favor of
long-term microbial methane formation in the Siljan crater, likely
fueled by thermogenic gas, seep oil and bitumen mobilized from
shales in the sedimentary successions and transported through
fracture conduits to the deeper granite aquifer. The sedimentary
successions, in turn, acted as cap rocks for the gas in the granite
fractures.
Input of hydrocarbons to the deep microbial communities has
potential to result in accumulation of methane in basement
fracture networks beneath sedimentary cap rocks. A relationship
between 13C-rich calcite and bitumen like at Siljan occurs in deep
crystalline rock fractures at Forsmark, Sweden25 and solid and
gaseous hydrocarbon occurrences of sedimentary origin occur in
fractured crystalline basement rocks on the British Isles63, Aus-
tralia64, and the United States65. Whereas microbial generation of
economic accumulations of methane within organic-rich shale
are known from several locations66, the extent of gas accumula-
tions in the upper crystalline continental crust buried beneath
sedimentary successions and in fractured impact structures are
less explored. The upper crystalline continental crust environ-
ment makes up one of the largest, but yet least surveyed, deep
biosphere habitats on Earth. The extent, continuity and physi-
cochemical prerequisites for gas accumulation here require more
attention in order to assess the significance of this underexplored
greenhouse gas source on a global scale.
In the Siljan impact structure, a relation between methane
cycling and deep subsurface life is evident. The physical influence
of the actual impact and the long-term effects are manifested by,
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first, abundant fracturing compared to surrounding rocks15 which
is particularly important in igneous lithologies where colonization
is restricted by the pore space2; second, dislocation of organic-rich
sedimentary rocks that provide pathways for surficial organics to
the deep endolithic communities; and third, development of a cap
rock enclosing methane at depth. These effects collectively enable
microbial colonization of the crater hundreds of millions of years
after the impact. Our findings of widespread long-lasting deep
microbial methane-forming communities in the Siljan crater
support the hypothesis that impact craters are favorable for deep
microbial colonization2,3. However, the link between microbial
methanogenesis and organics from Paleozoic shale challenges the
use of the Siljan crater as an analog for instantaneous extra-
terrestrial impact-related colonization. In an astrobiological con-
text, the Siljan crater findings are nevertheless of large importance,
as they display that multi-disciplinary micro-scale constraints for
microbial activity (stable isotopes, geochronology, biomarkers) are
needed to confirm that colonization and impact in ancient crater
systems are coeval. This is particularly important because post-
impact microbial colonization will likely occur in these favorable
deep microbial settings and can thus easily be misinterpreted as
impact related. Finally, the methods we have used here to provide
the first evidence of long-term microbial methane formation and
accumulation in a terrestrial impact crater would be optimal to
apply to other impact-crater fracture systems, including methane
emitting craters on Mars67, in order to enhance the understanding
of microbial activity and gas cycling in this underexplored
environment.
Methods
Materials. In this study, secondary calcite and pyrite coatings were collected
from open fractures in cores from a total of seven boreholes at Mora (n=5),
Solberga and Stumsnäs (Fig. 1) drilled in 2011–2018. Samples are from 64 to 642
m vertical depth from the ground surface. The co-genetic calcite and cubic pyrite
crystals occur on the fracture surfaces in the sedimentary rocks (Fig. 2a) and
crystalline basement (Fig. 2b–d). Calcite mainly occurs as subhedral flat aggre-
gates (Fig. 2i) to euhedral crystals of scalenohedral (Fig. 2e, f, m) or short c-axis
type (Fig. 2g). Polished cross-sections reveal growth zonation in several calcite
crystals (Fig. 2j–n). The paragenesis includes clay minerals, harmotome, apo-
phyllite, sphalerite, galena and quartz but most of these minerals are related to
the oldest growth phases of calcite. Seep oil and solid bitumen are present,
particularly in the sediments (e.g. at 326 and 382 depth, where oil covers and is
intermixed with calcite, Fig. 3a–d) but are also present in the crystalline base-
ment fractures (Fig. 3e–g). Additional samples were taken from fractured Upper
Ordovician Boda limestone in a quarry at Solberga. All boreholes are drilled
through an upper layer of Paleozoic sedimentary rocks into the Proterozoic
crystalline bedrock. The interface between the rock units is at between 250 to
406 m depth at Mora (borehole VM1: 250.9 m; VM2: 382m; 01–10C: 346 m;
01–11C: 400.5 m and CC1: 406 m) and Solberga (Solberga-1: 259 m). At
Stumsnäs, large scale impact-related faulting has caused a slab of Proterozoic
granite to overthrust the sedimentary successions which thus are sandwiched in
between blocks of granite at 196–286 m depth69,70. Samples were taken from
fractures in the sedimentary rocks (limestone and shale) and from the upper 212
m of the underlying crystalline basement. Sampling focused, but was not limited,
to borehole sections with elevated gas concentrations as observed during drilling.
The mineralogy and appearance of the uncoated fracture coatings were exam-
ined under low-vacuum conditions in a Hitachi S-3400N Scanning Electron
Microscope (SEM) equipped with an integrated energy dispersive spectroscopy
(EDS) system. The coatings were then scraped off for various analyses (fluid
inclusions, stable isotopes, radioisotopes and biomarkers).
Gas from isotube sampling and gases in waters from isojar sampling have been
analyzed by AB Igrene for chemical composition from a few deep borehole sections
(Supplementary Data 8).
Gas and oil seepageGas and oil seepage
Shale-thermogenic gas
Seep oil/gas/bitumen
Seep oil/gas
/bitumen
Seep oil/gas
/bitumen
Seep oil/gas/
bitumen
Biodegradation
Biodegradation
Microbial gas
formation
Ascending
mixed gas,
trapped under
caprock
Biodegradation
Microbial gas
formation
Seep oil/gas
/bitumen
Gas
trap
AOM
AOM
Shale-thermogenic gas
Seep oil/gas
/bitumen
Fault
Fault
Microbial gas
formation
Abiotic
gas ascent?
Paleozoic
sedimentary
successions
–350 m
–700 m
Proterozoic
crystalline
rocks
Seep oil/
gas/bitumen
Microbial gas
formation
Groundsurface
Seep oil/gas
/bitumen
Bio-
degradation
Shale-thermogenic gas
Shale-thermogenic gas
Gas
trap
Fig. 8 Conceptual model of the gas accumulation in the Siljan ring impact structure. Thermogenic gas formed in the (to varying degree) mature Silurian and
Ordovician black shales in the sedimentary strata. This gas and related seep oil and bitumen dispersed in the adjacent sedimentary successions with local
surficial seepage. Downward migration of these hydrocarbons has occurred into the granitic basement during fracture reactivation events. Biodegradation
of the hydrocarbons has occurred in the fracture system and secondary methane has formed in situ. The mixed gas, of microbial (dominantly) and
biodegraded thermogenic type, which also may have an abiotic end-member, has accumulated at the sedimentary-granite contact where anaerobic
oxidation of methane has occurred
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SIMS δ13C, δ18O, δ34S. Calcite and pyrite crystals were mounted in epoxy,
polished to expose cross-sections and examined with SEM to trace zonations and
impurities prior to SIMS analysis. Intra-crystal SIMS analysis (10 μm lateral beam
dimension, 1–2μm depth dimension) of sulfur isotopes in pyrite and carbon and
oxygen isotopes in calcite was performed on a Cameca IMS1280 ion microprobe at
NordSIM, Swedish Museum of Natural History, Stockholm, following the analy-
tical settings and tuning reported previously28. Sulfur was sputtered using a 133Cs+
primary beam with 20 kV incident energy (10kV primary, −10 kV secondary) and
a primary beam current of ~1.5 nA. A normal incidence electron gun was used for
charge compensation. Analyses were performed in automated sequences, with each
analysis comprising a 70 s pre-sputter to remove the gold coating over a rastered
15 × 15 µm area, centering of the secondary beam in the field aperture to correct for
small variations in surface relief and data acquisition in sixteen four second inte-
gration cycles. The magnetic field was locked at the beginning of the session using
an NMR field sensor. Secondary ion signals for 32S and 34S were detected simul-
taneously using two Faraday detectors with a common mass resolution of 4860 (M/
ΔM). Data were normalized for instrumental mass fractionation using matrix
matched reference materials which were mounted together with the sample mounts
and analyzed after every sixth sample analysis. Results are reported as per mil (‰)
δ34S based on the Canon Diablo Troilite (V-CDT)-reference value. Analytical
transects of up to seven spots were made from core to rim in the crystals. Up to
seventeen crystals were analyzed from each fracture sample. In total, 443 analyses
were made for δ34S(
34S/32S) of pyrite from 115 crystals from nineteen fracture
samples. The pyrite reference material S0302A with a conventionally determined
value of 0.0 ± 0.2‰(R. Stern, University of Alberta, pers. comm.) was used. Typical
precision on a single δ34S value, after propagating the within run and external
uncertainties from the reference material measurements was ±0.10‰.
For calcite, a total number of 984 δ13C (17 for δ18O) SIMS analyses were
performed. Settings follow those described for S isotopes, with some differences: O
was measured on two Faraday cages (FC) at mass resolution 2500, C used a FC/EM
combination with mass resolution 2500 on the 12C peak and 4000 on the 13C peak
to resolve it from 12C1H. Calcite results are reported as per mil (‰)δ13C based on
the Pee Dee Belemnite (V-PDB) reference value. Analyses were carried out running
blocks of six unknowns bracketed by two standards. Analytical transects of up to
nine spots were made from core to rim in the crystals. Up to fifteen crystals were
analyzed from each fracture sample. Analyses were made for 331 crystals from 67
fracture samples (50 from granite and 17 from sedimentary rock). Isotope data
from calcite were normalized using calcite reference material S0161 from a
granulite facies marble in the Adirondack Mountains, kindly provided by R.A.
Stern (Univ. of Alberta). The values used for IMF correction were determined by
conventional stable isotope mass spectrometry at Stockholm University on ten
separate pieces, yielding δ13C=−0.22 ± 0.11‰V-PDB (1 std. dev.) and δ18O=
−5.62 ± 0.11‰V-PDB (1 std. dev.). Precision was δ18O: ± 0.2–0.3‰and δ13C: ±
0.4–0.5‰. Values of the reference material measurements are listed together with
the samples in Supplementary Data 1 (C and O); Supplementary Data 2 (S).
LA-ICP-MS U-Pb. U-Pb geochronology via the in situ LA-ICP-MS method was
conducted at the Geochronology & Tracers Facility, NERC Isotope Geosciences
Laboratory (Nottingham, UK). The method utilizes a New Wave Research 193UC
excimer laser ablation system, coupled to a Nu Instruments Attom single-collector
sector-field ICP-MS. The method follows that previously described in Roberts
et al.71, and involves a standard-sample bracketing with normalization to NIST
614 silicate glass72 for Pb-Pb ratios and WC-1 carbonate71 for U-Pb ratios. The
laser parameters comprise a 100 μm static spot, fired at 10 Hz, with a ~8 J/cm2
fluence, for 30 s of ablation. Material is pre-ablated to clean the sample site with
150 μm spots for 3 s. NIST 614 is used for normalization of 207Pb/206Pb ratios. No
common lead correction is made; ages are determined by regression and the lower
intercept on a Tera-Wasserburg plot (using Isoplot 4.15). Duff Brown, a carbonate
previously measured by Isotope Dilution mass spectrometry was used as a vali-
dation, and pooling of all sessions yields a lower intercept age of 64.2 ± 1.6 Ma
(MSWD =4.0), overlapping the published age of 64.04 ± 0.67 Ma73. All ages are
plotted and quoted at 2σand include propagation of systematic uncertainties
according to the protocol described in Horstwood et al.74. Data are screened for
low Pb and low U counts below detection, and very large uncertainties on the Pb-
Pb and Pb-U ratios which indicate mixed analyses. The spots are also checked after
ablation for consistent ablation pit shape, and data are rejected if the ablations were
anomalous (this results from material cleaving off, or clipping the resin mount).
Eight samples of calcite were screened from the Siljan drill cores. Seven samples
yielded variably robust U-Pb ages. The other sample did not yield any determinable
single age populations that form a regression between common and radiogenic lead
compositions. The uranium contents of the samples are variable, as are the initial µ
(238U/204Pb) values. Along with the requirement of a closed isotopic system, i.e.
non-disturbed, the µ values have a large control on the likely success of resolving a
precise age, as they dictate the ratio of radiogenic to common lead that may exist in
the sample. The samples were measured on one or two occasions, both as small
mounted chips, and on larger chips that were previously used for in situ stable
isotope analysis. The data do not reflect simple mixing between common and
radiogenic lead, but across the grains represent either different age components,
variable common lead compositions, and/or disturbed isotopic systematics. The
radiogenic data that provide the ages discussed in this manuscript are from pristine
calcite and are interpreted to represent a primary age of this calcite growth. Results
are of mixed quality (i.e. both low and high MSWDs), indicating minor open
system behavior, and/or mixing between domains of different age for the samples
with high MSWD. The interpreted lower intercept ages for the latter are based on
radiogenic data and are still useful for broad age interpretation. Full analytical data
from the sessions are listed in Supplementary Data 4; ages in Supplementary
Data 5; analytical conditions in Supplementary Data 6).
LA-MC-ICP-MS 87Sr/86Sr. The 87Sr/86Sr values of the calcite crystals were
determined by LA-MC-ICP-MS analysis at the Vegacenter, Swedish Museum of
Natural History, Stockholm, using a Nu plasma (II) MC-ICP-MS, and an ESI
NWR193 ArF eximer laser ablation system. Ablation frequency was 15 Hz, spot
size 80 μm and fluence 2.8 J/cm2and the same crystal growth zones analyzed with
SIMS for δ13C were targeted. Wash-out and ablation times were both 45 s. The
87Sr/86Sr analyses were normalized to an in-house brachiopod reference material
‘Ecnomiosa gerda’(linear drift and accuracy correction) using a value established
by TIMS of 0.709181 (2sd 0.00000475). A modern oyster shell from Western
Australia was used as a secondary reference material and analyzed at regular
intervals together with the primary reference. The accuracy of these analyses was
quantified by comparison to the modern seawater value for 87Sr/86Sr of 0.7091792
± 0.000002176. Values of the reference material measurements are listed in Sup-
plementary Data 1.
GC-MS, biomarkers. The mineral coating samples (calcite-dominated) were gently
flushed with acetone to remove surface contaminations and then ground with an
agate pistil. The sample powders were first extracted with 2 mL of pre-distilled
dichloromethane/methanol in Teflon-capped glass vials (ultrasonication, 15 min,
40 °C). The supernatant was decanted after centrifuging. Extraction was repeated
two times, with dichloromethane and with hexane as solvents. After evaporation of
the combined extracts and re-dissolution in pure dichloromethane, the solvents
were dried with N
2
. The total organic extract (TOE) was derivatized by adding 100
μL BSTFA (60 °C, 1.5 h). The sample was dried with N
2
, and mobilized with 100 μl
n-hexane and stored at –18 °C until measurement. The sample remnants were
dissolved and demineralized by adding 5 ml of TMCS/Methanol (1 +9) for 12 h
and then derivatised for 90 min at 80 °C. After cooling the samples were mixed
with n-hexane and the supernatants were decanted and collected separately. This
procedure was repeated three times. The combined supernatants were dried with
nitrogen and remobilized with 100 µl n-hexane. 1 μL of each sample extract was
analyzed via on-column injection into a Varian CP-3800 GC/1200-quadrupole MS
(70 keV) equipped with a fused silica column (Phenomenex ZB-5; 30 m length,
0.32 μm inner diameter; 0.25 μmfilm thickness). The GC oven was programmed
from 80 °C (held 3 min) to 325˚C (held 40 min) at 6 °C/min. He was used as carrier
gas at 1.4 ml/min. Compounds were assigned by comparison with published mass
spectral data.
ToF-SIMS. Right before ToF-SIMS analyzes, the rock containing fractures with
13C-rich calcite coatings was cracked open, using clean tweezers (heptane, acetone
and ethanol in that order), to expose fresh surfaces. This sample was taken from a
newly drilled cored borehole. Small pieces of rock containing putative organic
remains, and aliquots of dominantly putative organic material, were then mounted
with clean tweezers on double-sticky tape on a silica wafer. The ToF-SIMS analysis
was performed on a ToF-SIMS IV (ION-TOF GmbH), at RISE, Sweden, by ras-
tering a 25 keV Bi
3
+beam (pulsed current of 0.1 pA) over an area of ~200 × 200
µm for 200–300 s. The analyzes were performed in positive and negative mode at
high mass resolution (bunched mode: Δl ~ 3 µm, m⁄Δm ~ 2000–4000 at m⁄z 30).
An electron gun was used for charge composition. As a control, additional spectra
were also acquired from the tape to confirm that samples had not been con-
taminated by the tape.
Fluid inclusions. Fluid inclusions were studied using microthermometry techni-
ques for handpicked calcite crystals (0.5–1.5 mm in size). A conventional micro-
scope was used to get an outlook of the samples and the distribution of the fluid
inclusions. Microthermometric analyses of fluid inclusions were made with a
Linkam THM 600 stage mounted on a Nikon microscope utilizing a ×40 long
working-distance objective. The working range of the stage is from −196 to +600 °
C. The thermocouple readings were calibrated by means of SynFlinc synthetic fluid
inclusions and well-defined natural inclusions in Alpine quartz. The reproducibility
was ± 0.1 °C for temperatures below 40 °C and ±0.5°C for temperatures above
40 °C.
Gas analysis. Gases were collected by AB Igrene from boreholes VM2 and VM5 by
isotube sampling (and additional isojar sampling of water from the VM2 borehole)
and analyzed at commercial laboratory Applied Petroleum Technology AS, Nor-
way. Selected values were extracted from AB Igrene’s database for the present work.
All laboratory procedures follow NIGOGA, 4th Edition.
Aliquots for GC analysis of gas components of the samples were transferred to
exetainers. 0.1–1 ml were sampled using a Gerstel MPS2 autosampler and injected
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Content courtesy of Springer Nature, terms of use apply. Rights reserved
into a Agilent 7890 RGA GC equipped with Molsieve and Poraplot Q columns and
aflame ionization detector.
The δ13C composition of the hydrocarbon gas components was determined by a
GC-C-IRMS system. Aliquots were sampled with a syringe and analyzed on a Trace
GC2000, equipped with a Poraplot Q column, connected to a Delta plus XP IRMS.
The components were burnt to CO
2
and water in a 1000 °C furnace over Cu/Ni/Pt .
The water was removed by Nafion membrane separation. Repeated analyses of
standards indicate reproducibility better than 1‰PDB (2 sigma).
The δ13C
CH4
values of low CH
4
concentrations were determined by a Precon-
IRMS system. Aliquots were sampled with a GCPal autosampler. CO
2
, CO and
water were removed on chemical traps. Other hydrocarbons than CH
4
and
remaining traces of CO
2
were removed by cryotrapping. The CH
4
was burnt to
CO
2
and water in a 1000 °C furnace over Cu/Ni/Pt. The water was removed by
Nafion membrane separation. The sample preparation system (Precon) was
connected to a Delta plus XP IRMS for δ13C analysis. Repeated analyses of
standards indicate reproducibility better than 1‰PDB (2 sigma).
The δ2H
CH4
isotopic composition of methane was determined by a GC-C-IRMS
system. Aliquots were sampled with a GCPal and analyzed on a Trace GC2000,
equipped with a Poraplot Q column, connected to a Delta plus XP IRMS. The
components were decomposed to H
2
and coke in a 1400 °C furnace. The
international standard NGS-2 and an in-house standard were used for testing
accuracy and precision. Repeated analyses of standards indicate reproducibility
better than 10‰PDB (2 sigma).
Data availability
All relevant data are included in the Supplementary material to this article. AB Igrene
owns the gas data, and for this study selected data have been extracted from their
database (placed in Supplementary Data 8).
Received: 30 January 2019; Accepted: 27 September 2019;
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Acknowledgements
Thanks to AB Igrene for access to drill cores, drilling logs and gas data. Swedish research
council (contract 2017–05186 to H.D., 2015–04129 to S.S., 2017–04129 to M.I.) and Formas
(contract 2017–00766 to H.D. and M.W.) are thanked for financial support. K. Lindén,
M. Tillberg, and M. Schmitt are thanked for analyticalor sample preparation assistance, and
University of Gothenburg for access to SEM. This is NordSIM publication 617 and
Vegacenter publication 20. Open access funding provided by Linnaeus University.
Author contributions
H.D. initiated and planned the study, carried out sampling, sample preparation, SEM-,
SIMS- and LA-MC-ICP-MS analyzes, did the conceptual modeling and wrote the paper.
N.R. carried out U-Pb geochronology and data reduction. C.H. carried out biomarker
analyzes and interpretation, M.W. handled the SIMS-equipment, instrument tuning and
data reduction, S.S. carried out the ToF-SIMS analyzes and data reduction. E.K. handled
the LA-MC-ICP-MS-equipment, C.B. carried out fluid inclusion analysis. M.I. and M.Å.
were involved in writing.
Competing interests
The authors declare no competing interests.
Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41467-
019-12728-y.
Correspondence and requests for materials should be addressed to H.D.
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