Unravelling abiogenic and biogenic sources of methane in the
Earth’s deep subsurface
B. Sherwood Lollar
a,
*, G. Lacrampe-Couloume
a
,
1
, G.F. Slater
a
,
1
, J. Ward
a
,
1
,
D.P. Moser
b
,
2
, T.M. Gihring
b
,
3
, L.-H. Lin
c
,
4
, T.C. Onstott
c
,
4
a
Department of Geology, 22 Russell St., University of Toronto, Toronto, Ontario, Canada M5S 3B1
b
Environmental Microbiology Group, Pacific Northwest National Laboratory, Richland WA 99352, USA
c
Dept. of Geosciences, Guyot Hall, Princeton University, Princeton NJ 08544, USA
Accepted 5 September 2005
Abstract
At four underground sites in Precambrian Shield rocks in Canada and South Africa, hydrocarbon and hydrogen gases exsolving
from saline fracture waters are analyzed for compositional and isotopic signatures. Dominated by reduced gases such as CH
4
, H
2
and higher hydrocarbons (ethane, propane, butane), the most
13
C-enriched methane end-members at all four sites show a pattern of
carbon and hydrogen isotopic values similar to abiogenic gases produced by water–rock interaction that have been identified
previously at one site on the Precambrian Shield in Canada. The abiogenic nature of these gases was not previously recognized due
to mixing with a second methane component produced by microbial processes. The microbial methane end-member is identified
based on carbon and hydrogen isotopic signatures, and DNA gene amplification (PCR) data that indicate the presence of
methanogens. A framework is presented to estimate the relative contribution of abiogenic versus microbial hydrocarbon gases
at these sites. This approach has important implications for evaluation of potential abiogenic hydrocarbon reservoirs in a wide
range of geologic settings, including the longstanding controversy concerning the possible contribution of abiogenic gases to
economic petroleum hydrocarbon reservoirs. The association of high concentrations of H
2
with
13
C-enriched CH
4
end-members,
and H
2
depletion in the
13
C-depleted methanogenic end-members further suggests the possibility that abiogenic gases may support
H
2
autotrophy linked to methanogenesis in the deep subsurface.
D
2005 Elsevier B.V. All rights reserved.
Keywords:
Abiogenic; Methane; Hydrogen; Autotrophy; Deep biosphere; Mars
0009-2541/$ - see front matter
D
2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.chemgeo.2005.09.027
* Corresponding author. Tel.: +1 416 978 0770; fax: +1 416 978 3938.
E-mail addresses:
bslollar@chem.utoronto.ca (B. Sherwood Lollar), glc@geology.utoronto.ca (G. Lacrampe-Couloume), gslater@mcmaster.ca
(G.F. Slater), ward@geology.utoronto.ca (J. Ward), duane.moser@dri.edu (D.P. Moser), gihring@ocean.fsu.edu (T.M. Gihring), lhlin@ntu.edu.tw
(L.-H. Lin), tullis@princeton.edu (T.C. Onstott).
1
Fax: +1 416 978 3938.
2
Fax: +1 702 862 5360.
3
Fax: +1 850 644 2581.
4
Fax: +1 609 258 1274.
Chemical Geology 226 (2006) 328 – 339
www.elsevier.com/locate/chemgeo
1. Introduction
Recent reports of CH
4
in the Mars atmosphere fo-
cussed scientific and public attention on possible geo-
logical and biological sources of these hydrocarbons
(
Formisano et al., 2004; Kerr, 2004
). Resolving the
question of the origin of atmospheric CH
4
on Mars is
even more challenging given that distinguishing abio-
genic versus biogenic sources of CH
4
in the terrestrial
subsurface is still controversial (
Gold, 1979; Kenney et
al., 2002; Shock, 1995
). Deep subsurface fluids in
Precambrian Shield rocks have been shown to be dom-
inated by reduced gases such as CH
4
and locally, high
concentrations of H
2
(up to 30% by volume) (
Sherwood
Lollar et al., 1993a,b
).
Sherwood Lollar et al. (2002)
used stable isotope signatures to suggest that CH
4
and
higher hydrocarbon gases (ethane, propane and butane)
at Kidd Creek mine on the Canadian Shield are pro-
duced abiogenically by water–rock interaction such as
surface-catalysed polymerization (
Anderson, 1984;
Foustoukos and Seyfried, 2004
); metamorphism of
graphite–carbonate bearing rocks (
Giardini and Salotti,
1969; Holloway, 1984; Kenney et al., 2002
); and other
gas–water–rock alteration reactions such as serpentini-
zation (
Berndt et al., 1996; Charlou and Donval, 1993;
Horita and Berndt, 1999; Kelley et al., 2001, 2005;
McCollom and Seewald, 2001; Vanko and Stakes,
1991
). In this paper, data from 4 new sites in Canada
and South Africa suggests that abiogenic hydrocarbon
gases are more globally pervasive than has been under-
stood previously. This is due to the fact that at many
sites, the distinct abiogenic signature of such hydrocar-
bons is obscured by mixing with microbial CH
4
. Based
on this data, we present a model for identification of
abiogenic hydrocarbons through the resolution of mi-
crobial and abiogenic mixing — an approach that is
applicable to a wide variety of crustal settings where
potential abiogenic hydrocarbon reserves have been
suggested (
Gold, 1979; Kenney et al., 2002; Shock,
1995
).
2. Geological setting and samples
In gold and base-metal mines throughout the Pre-
cambrian Shield rocks of Canada, Finland and South
Africa, flammable gases discharge from fractures and
exploration boreholes (
Nurmi and Kukkonen, 1986;
Lahermo and Lampen, 1987; Cook, 1998; Sherwood
Lollar et al., 1993a,b
). Originally dissolved in saline
groundwater (TDS levels range from several 1000 to
tens of 1000s of ppm) in sealed fracture systems in the
rocks, gases are released via depressurization into mine
workings at rates of 1 to
N
30 L gas/min/borehole
(
Sherwood Lollar et al., 1993a,b
). The groundwaters
are typically NaCaCl-rich or CaNaCl-rich with low
levels of SO
4
(typically
b
15 ppm and always
b
100
ppm) with pH levels between 7–9. This study focuses
on two sites in Canada (Kidd Creek and Copper Cliff
South mines) and three in South Africa (Driefontein,
Kloof and Mponeng mines). Kidd Creek mine, situated
in the southern volcanic zone of the Abitibi greenstone
belt (approximately 2700 Ma), 24 km N of Timmins,
Ontario is one of the world’s largest volcanogenic
massive sulfide deposits. All samples were collected
from 2–2.1 kmbls (kilometers below land surface). The
Cu–Ni ore at Copper Cliff South mine (CCS) in Sud-
bury, Ontario is associated with a quartz diorite dike
offset of the Sudbury Igneous Complex — a micro-
pegmatite, norite and quartz diorite irruptive emplaced
approximately 1840 Ma (
Cochrane, 1984
) and samples
were from approximately 1.3 kmbls. In South Africa,
Kloof, Driefontein and Mponeng mines are all located
west of Johannesburg in the Witwatersrand Basin — a
large Archean intracratonic basin composed of volca-
nosedimentary sequences that unconformably overlie
3.0 Ga granitic basement. The basin is divided chrono-
logically into quartzite, shale and minor volcanic strata
of the Witwatersrand Supergroup (2900 Ma), the basal-
tic lava sequence of the Ventersdorp Supergroup (2700
Ma) and the overlying clastic and dolomitic sediments
of the Transvaal Supergroup (2400–2500 Ma) (
Coward
et al., 1995
). Almost all the samples in this study are
from the Ventersdorp Supergroup and are located be-
tween 2.7 and 3.4 kmbls. The exceptions are borehole
DR938H3, which while drilled in the Ventersdorp,
intersects the underlying Witwatersrand Supergroup at
approximately half its 750 m length, and DR9IPC,
which is drilled in the overlying Transvaal Supergroup
and is located at 0.9 kmbls.
3. Methodology
3.1. Sampling methods
All gas and fracture water samples were collected at
the borehole collar after the method of
Sherwood Lollar
et al. (2002)
and
Ward et al. (2004)
. A packer was
placed into the opening of the borehole and sealed to
the inner rock walls below water level to seal the
borehole from the mine air and minimize air contami-
nation. Gas and water were allowed to flow through the
apparatus long enough to displace any air remaining in
the borehole or the apparatus before sampling. Plastic
tubing was attached to the end of the packer and the
B. Sherwood Lollar et al. / Chemical Geology 226 (2006) 328–339
329
flow of gas and/or water from the borehole was directed
into an inverted graduated funnel. Gases collected in
the inverted funnel were transferred directly into evac-
uated vials through a needle that was attached to the top
of the funnel. The gas sampling vials were pre-evacu-
ated 130 ml borosilicate vials sealed with butyl blue
rubber stoppers prepared after the method of
Oremland
and Des Marais (1983)
. Vials were pre-fixed with 50
A
l of a saturated HgCl
2
solution to kill any microbes
contained in the sample so microbial activity post-sam-
pling would not alter the gas composition and isotopic
signatures. Previous studies, comparing the isotopic
values of gases taken at the borehole collars to values
determined for gases in solution at depth in the same
boreholes, showed that exsolution of the hydrocarbon
gases from solution does not alter their isotopic signa-
tures (
Sherwood Lollar et al., 1993a,b, 1994
).
For the South African sites, the packer was sterilized
by autoclaving before insertion into the borehole in
order to reduce the possibility of introducing surface
microrganisms by that means. In mining environments,
aseptic drilling techniques were not possible. Microbial
populations were characterized and compared however
between the saline fracture waters from these sites, the
low salinity mine service water, and ventilation air.
Comparison of 16S rRNA gene clone libraries from
these possible sources of contamination and from the
fracture waters yielded no commonalities (
Kieft et al.,
1999; Onstott et al., 2003; Takai et al., 2001
). In
addition the fact that dissimilatory sulphate reductase
(DSR) and 16S ribosomal (rRNA) genes amplified
from the fracture water DNA yield sequences that are
most similar to those reported for sulphate-reducing
bacteria isolated from other subsurface sites is consis-
tent with the fracture water microbial populations being
in situ subsurface communities and not surface con-
taminants (
Baker et al., 2003
).
3.2. Compositional gas analysis
Compositional analyses of gas samples were per-
formed at the Stable Isotope Laboratory at the Univer-
sity of Toronto. A Varian 3400 GC equipped with a
flame ionization detector (FID) was used to determine
concentrations of CH
4
, C
2
H
6
, C
3
H
8
and C
4
H
10
. The
hydrocarbons were separated on a J and W Scientific
GS-Q column (30 m
0.32 mm ID) with a helium gas
flow and temperature program: initial 60
8
C hold 2.5
min, increase to 120
8
C at 5
8
C/min. A Varian 3800 GC
equipped with a micro-thermal conductivity detector
(
A
TCD) and a Varian Molecular Sieve 5A PLOT col-
umn (25 m
0.53 mm ID) was used to determine
concentrations of the inorganic gas components (H
2
,
He, Ar, O
2
, CO
2
and N
2
). To determine concentrations
of Ar, O
2
and N
2
the helium gas flow rate was 3 ml/min
and the temperature program was: initial 30
8
C hold 6
min, increase to 80
8
C at 15
8
C/min, hold 4 min. To
determine CO
2
concentrations, the helium gas flow rate
was 50 ml/min and temperature program was: initial 60
8
C, increase to 250
8
C at 20
8
C/min, hold 6 min. To
determine concentrations of H
2
and He, the argon
carrier gas flow rate was 2 ml/min and temperature
program was: initial 10
8
C hold 10 min, increase to
80
8
C at 25
8
C/min, hold 7 min. All analyses were run
in triplicate and mean values are reported in
Table 1
.
Reproducibility for triplicate analyses was
F
5%.
3.3. Isotopic analysis
Stable carbon and hydrogen isotopic analysis for all
hydrocarbons were performed at the University of Tor-
onto (
Table 2
). Analyses for
d
13
C values were per-
formed by continuous flow compound specific carbon
isotope ratio mass spectrometry with a Finnigan MAT
252 mass spectrometer interfaced with a Varian 3400
capillary GC. Hydrocarbons were separated by a Por-
aplot Q
k
column (25 m
0.32 mm ID) with temper-
ature program: initial 40
8
C hold 1 min, increase to 190
8
C at 5
8
C/min, hold 5 min. To separate CO
2
from CH
4
the program was started at 10
8
C (hold 2 min) increased
to 190
8
C at 5
8
C/min (hold 5 min). Total error incor-
porating both accuracy and reproducibility is
F
0.5
x
with respect to V-PDB standard.
The
d
2
H analysis was performed on a continuous
flow compound specific hydrogen isotope mass spec-
trometer which consists of an HP 6890 gas chromato-
graph (GC) interfaced with a micropyrolysis furnace
(1465
8
C) in line with a Finnigan MAT Delta
+
-XL
isotope ratio mass spectrometer. The hydrocarbon
gases were separated on a Poraplot Q
k
column (25
m
0.32 mm ID) with a helium carrier at 2.2 ml/min
and temperature program: initial 35
8
C hold 3 min,
increase to 180
8
C at 15
8
C/min. Total error incorpo-
rating both accuracy and reproducibility is
F
5
x
with
respect to V-SMOW.
3.4. DNA gene amplification (PCR)
Methyl coenzyme M reductase
a
-subunit genes
(
mcrA
) were amplified from community DNA extracts
according to the methods of
Hales et al. (1996)
. Ar-
chaeal 16S rRNA gene was amplified using a nested
PCR reaction in which PCR product generated using
the primers 21F (
Moyer et al., 1998
), and 1492R (
Rey-
B. Sherwood Lollar et al. / Chemical Geology 226 (2006) 328–339
330
senbach et al., 2000
) was used as template in PCR
reactions with the
b
nested
Q
primers 21F and 958R
(
Moyer et al., 1998
).
McrA
and archaeal 16S rRNA
gene PCR products were cloned using Invitrogen
TOPO kits. For each library, 48 clones were screened
by restriction fragment length polymorphism. Unique
clones were sequenced, then analyzed using BLAST
(
Altschul et al., 1997
) to identify sequences in the
NCBI Genbank database having high similarity (
Table
3
). Sequence identities were calculated using BioEdit.
4. Results and discussion
4.1. Isotopic patterns suggest an abiogenic origin
Mantle-derived abiogenic hydrocarbons are typically
identified based on three criteria: a
d
13
C value for CH
4
more enriched than
25
x
; a
b
carbon isotopic reversal
Q
trend of increasing isotopic depletion in
13
C with in-
creasing molecular weight for CH
4
–ethane–propane–
butane; and a
3
He /
4
He ratio indicative of mantle-de-
rived helium (
R
/ Ra
N
0.1) (
Jenden et al., 1993
). Based
on these criteria, coal bed or thermogenic hydrocarbon
gases with anomalously enriched
d
13
C values and/or
anomalously depleted
d
2
H values for methane could be
distinguished from mantle-derived abiogenic gases
(
Jenden et al., 1988; Jenden and Kaplan, 1989; Poreda
et al., 1986, 1988; Lyon and Hulston, 1984; Rigby and
Smith, 1981
). Over the past decade however, there has
been a growing body of literature that indicates that not
all abiogenic gases are mantle-derived. A variety of low
temperature water–rock interactions have been shown
to produce both CH
4
and higher hydrocarbons such as
ethane, propane, and butane — including surface-cata-
lyzed polymerization from reduction of CO or CO
2
in a
Fischer–Tropsch synthesis; heating or metamorphism
of graphite or carbonate bearing rocks; or other
vapor–water–rock alteration reactions such as serpenti-
nization (
Holloway, 1984; Yuen et al., 1990; Berndt et
al., 1996; Hu et al., 1998; Horita and Berndt, 1999;
McCollom and Seewald, 2001; Foustoukos and Sey-
fried, 2004
). Significantly, several of these studies have
demonstrated that production of abiogenic CH
4
by
these water–rock interactions can result in
d
13
C values
as depleted as
57
x
(
Yuen et al., 1990; Hu et al.,
1998; Horita and Berndt, 1999
). In a crustal-dominated
geologic setting, these processes will produce abiogenic
hydrocarbons whose
d
13
C values reflect local crustal
carbon sources and will not have either
13
C-enriched
d
13
C values or
R
/ Ra values
N
0.1. In addition, it has
Table 1
Gas compositional data (in %)
Site
Borehole No.
Ar
H
2
He
O
2
N
2
CH
4
C
2
H
6
C
3
H
8
C
4
H
10
Copper Cliff
CCS4577
0.06
54.0
3.46
0.11
1.92
34.4
5.31
0.49
0.11
Copper Cliff
CCS4547
0.07
43.0
3.38
0.12
2.20
38.1
5.28
0.56
0.12
Copper Cliff
CCS4546
0.17
9.94
4.37
0.52
6.38
69.5
7.14
0.78
0.17
Copper Cliff
CCS4572
0.05
57.8
2.62
0.15
1.72
25.3
4.16
0.38
0.09
Copper Cliff
CCS4880
0.27
19.7
6.42
0.10
4.54
59.3
7.39
1.11
0.29
Mponeng
MPA
NA
3.30
9.06
4.77
28.1
53.8
2.47
0.36
0.08
Mponeng
MP104
NA
11.5
12.3
3.65
21.1
49.6
3.99
0.60
0.13
Driefontein
DR548
NA
10.3
3.05
6.18
23.6
50.7
3.85
0.52
0.11
Driefontein
DR938H1
NA
b
0.01
0.90
16.5
72.5
5.45
0.14
0.02
b
0.01
Driefontein
DR938H3-0m
NA
0.74
5.98
0.55
14.0
76.0
3.15
0.32
0.06
Driefontein
DR938H3
V
-0m*
NA
0.32
4.64
4.95
28.6
61.4
2.45
0.26
0.04
Driefontein
DR938H3-648 m
NA
NA
NA
NA
NA
NA
NA
NA
NA
Driefontein
DR938CH1
NA
b
0.05
0.21
23.4
89.9
1.19
b
0.05
b
0.05
b
0.05
Driefontein
DR9IPC
NA
b
0.05
2.75
3.77
81.9
11.6
b
0.05
b
0.05
b
0.05
Kloof
KL1GH
NA
9.15
3.42
14.5
56.4
10.7
0.49
b
0.01
b
0.01
Kloof
KL441H3
NA
b
0.01
15.9
0.43
45.1
33.0
0.66
0.07
0.02
Kloof
KL739
NA
9.25
13.5
b
0.04
7.72
64.9
2.86
0.41
0.08
Kloof
KL441H2
1.68
b
0.01
15.6
6.55
61.9
23.4
0.51
0.05
b
0.01
Kloof
KL443HWND1
1.39
b
0.01
18.2
1.24
13.3
57.3
2.03
0.28
0.05
Kloof
KL443HWDN
1.79
b
0.01
20.8
0.97
16.8
53.4
2.04
0.21
0.03
a. All CO
2
was below detection limit.
b. NA — not analyzed.
c. C
4
H
10
incorporates both
n
-butane and
iso
-butane.
d. *DR938H3
V
-0 m is a sample from the same borehole as DR938H3-0 m taken 8 months afterwards. Such temporal sampling was not possible for
the other boreholes due to the common practice of sealing them immediately after completion of drilling.
e. DR938H3-0 m and DR938H3-648 m were collected from the same borehole on the same day. The 0 m sample was taken at the borehole collar
while the 648 m sample was taken at 648 m depth.
B. Sherwood Lollar et al. / Chemical Geology 226 (2006) 328–339
331
become clear that the
b
carbon isotopic reversal
Q
trend
alone is not sufficient evidence to support an abiogenic
origin (
Horita and Berndt, 1999
).
Based on crustal
3
He /
4
He ratios at all sites, no
mantle-derived component for the Precambrian Shield
hydrocarbon gases is indicated (
Lippmann et al., 2003;
Sherwood Lollar et al., 1993b
).
Sherwood Lollar et al.
(2002)
suggested a crustal abiogenic origin due to
water–rock interactions for the hydrocarbon gases at
Kidd Creek however, based on a new criteria — an
inverse correlation of
13
C-depletion and
2
H-enrichment
between CH
4
and ethane (
Fig. 1
A). Building on a
hypothesis for carbon isotope distribution patterns first
proposed by
Des Marais et al. (1981)
, it was suggested
that this pattern results from synthesis of higher molec-
ular weight hydrocarbons from CH
4
during polymeriza-
tion. In such a process
12
CH
4
reacts faster than
13
CH
4
to
form chains, so that
12
C is more likely to be incorporated
into larger hydrocarbon chains in polymerization reac-
tions, whereas, owing to preferential cleavage of the
weaker
12
C–
1
H bond versus the
12
C–
2
H bond, the
light (
1
H) isotope will be preferentially eliminated
(
Sherwood Lollar et al., 2002
). In contrast, the pattern
for commercial thermogenic natural gas deposits con-
sists of a positive correlation of
d
13
C and
d
2
H values
between CH
4
, ethane and the higher hydrocarbon gases
propane and butane (
Fig. 1
A). This isotopic pattern of
increasing isotopic enrichment in both
13
C and
2
H with
increasing molecular weight for the C
1
–C
4
homologues
for thermogenic gases results from production of hydro-
carbons by thermal cracking of a high molecular weight
organic precursor (
Des Marais et al., 1981; Schoell,
1988
). At each of the 4 newly investigated Precambrian
Shield sites in Canada and South Africa from this study,
hydrocarbon gases were identified with the same pattern
of
13
C-depletion and
2
H-enrichment between CH
4
and
Table 3
16S rRNA gene and
mcrA
clones from borehole DR9IPC having high relatedness to known methanogenic organisms
DR9IPC clone (Genbank accession number)
Percent of library
Nearest cultivated relative
Identity (%)
16S rRNA 1 (AY604061)
40.0
Methanobacterium curvum
93
mcrA 1 (AY604057)
23.7
Methanosarcina barkeri
99
mcrA 2 (AY604058)
73.7
Methanobacterium bryantii
86
mcrA 3 (AY604059)
2.7
Methanobacterium aarhusense
83
Table 2
Carbon and hydrogen isotopic values (in
x
) for CH
4
–C
4
hydrocarbon gases
Site
Borehole no.
d
13
C
CH
4
d
2
H
CH
4
d
13
C
C
2
d
2
H
C
2
d
13
C
C
3
d
2
H
C
3
d
13
C
n
C
4
d
2
H
n
C
4
Copper Cliff
CCS4577
36.1
413
24.6
266
ND
ND
ND
ND
Copper Cliff
CCS4547
34.4
424
28.1
290
ND
ND
ND
ND
Copper Cliff
CCS4546
32.0
452
34.6
348
33.4
ND
ND
ND
Copper Cliff
CCS4572
36.7
401
24.4
269
ND
ND
ND
ND
Copper Cliff
CCS4880
37.3
372
31.6
301
ND
ND
ND
ND
Mponeng
MPA
27.8
349
35.0
267
33.3
212
ND
ND
Mponeng
MP104
32.8
366
37.6
270
34.8
193
35.3
88
Driefontein
DR548
46.5
403
50.5
285
47.2
192
45.6
72
Driefontein
DR938H1
53.6
295
42.9
326
42.3
ND
ND
ND
Driefontein
DR938H3-0 m
40.2
368
43.1
290
42.0
ND
ND
ND
Driefontein
DR938H3
V
-0 m*
42.3
369
ND
ND
ND
ND
ND
ND
Driefontein
DR938H3-648 m
40.9
371
44.0
234
42.9
179
ND
ND
Dreifontein
DR938CH1
47.5
329
43.2
ND
ND
ND
ND
ND
Driefontein
DR9IPC
55.5
218
ND
ND
ND
ND
ND
ND
Kloof
KL1GH
29.2
312
33.2
230
ND
ND
ND
ND
Kloof
KL441H3
40.0
253
30.9
241
25.2
ND
ND
ND
Kloof
KL739
28.7
300
30.7
230
27.2
142
ND
ND
Kloof
KL441H2
37.8
257
32.9
198
34.0
105
ND
ND
Kloof
KL443HWND1
34.4
327
35.6
251
33.7
208
ND
ND
Kloof
KL443HWDN
34.3
329
34.3
269
33.5
206
33.6
207
a. ND — below detection limit for isotope analysis.
b. *DR938H3
V
-0m is a sample from the same borehole as DR938H3-0m taken 8 months afterwards. Such temporal sampling was not possible for
the other boreholes due to the common practice of sealing them immediately after completion of drilling.
c. DR938H3-0m and DR938H3-648m were collected from the same borehole on the same day. The 0 m sample was taken at the borehole collar
while the 648 m sample was taken at 648 m depth.
B. Sherwood Lollar et al. / Chemical Geology 226 (2006) 328–339
332
ethane as the proposed abiogenic gases from Kidd Creek
(
Fig. 1
B;
Table 2
). Interestingly, for each site only the
most
13
C-enriched samples show this pattern.
4.2. Mixing trends
Whereas the Kidd Creek data set is remarkably
uniform, one sample (KC7792) has a
d
13
C
CH4
value
significantly more depleted in
13
C and enriched in
2
H
than other samples from this site (
Fig. 1
A).
Sherwood
Lollar et al. (2002)
suggested that this might be due to
mixing with a more
13
C-depleted and
2
H-enriched mi-
crobial CH
4
component in this borehole. In the current
study, the hydrocarbon gas samples analysed for 4 new
Precambrian Shield sites show a much wider range of
CH
4
isotopic signatures than at Kidd Creek (
Figs. 1B
and 2
). In all cases, the proposed abiogenic signature
was observed only for the samples with the most
13
C-
enriched CH
4
values at each site (DR938H3 at both 0
and 685 m, DR548; KL739 and KL1GH; CCS4546;
and both samples from Mponeng — MP104 and MPA;
Fig. 1
B). The distinct
13
C–
2
H isotopic patterns between
CH
4
and ethane for these samples (
Fig. 1
B), as well as
their significantly depleted
d
2
H values compared to
typical thermogenic gases (
Fig. 2
), do not support a
thermogenic origin for these
13
C-enriched end-mem-
bers. Furthermore, as at Kidd Creek, the majority of
the data points from CCS, Driefontein and Kloof Mines
fall along linear trends between the most
13
C-enriched
and
2
H-depleted end-members, and samples with more
13
C-depleted and
2
H-enriched values (
Fig. 2
), raising
the possibility of mixing between these two end-mem-
bers. For CCS, Kloof and Kidd Creek, all data fall
along a single trend from the
13
C-enriched end-mem-
bers identified in
Fig. 1
B, whereas for Driefontein, two
possible trends can be drawn through the data, one for
DR938H3 and one for DR548. At Mponeng, both
samples MP104 and MPA are
13
C-enriched and show
the proposed abiogenic signature (
Fig. 1
B) and this is
consistent with the lack of any mixing trend in
Fig. 2
.
In
Fig. 2
, solid lines indicate the conventionally ac-
cepted fields of
d
13
C and
d
2
H values for microbial and
thermogenic CH
4
based on empirical studies (
Schoell,
1988
). The hatched trend lines indicate that
13
C-depleted
and
2
H-enriched end-members at these sites could theo-
retically be either thermogenic or microbial in origin
based on the distribution of the
d
13
C and
d
2
H fields.
Other lines of evidence indicate that this end-member is
microbial however, and suggest that microbial CH
4
mix-
ing with abiogenic gases produces the observed trend
lines. For borehole DR9IPC, 16S rRNA gene cloning
and
mcrA
(methyl coenzyme M reductase) gene cloning
indicate the presence of methanogens (
Table 3
). In addi-
tion, the
d
13
C and
d
2
H values for DR9IPC are similar to
the values for microbial CH
4
identified in several other
sites in the Witwatersrand Basin (
Ward et al., 2004
). In
particular, fracture water from Masimong and Merrie-
spruit, located 150 km southwest of Driefontein, have
d
13
C and
d
2
H values for CH
4
of
60.7 and
207
x
(MM5); and
53.7 and
194
x
(MR1), respectively
(
Fig. 2
). The microbial origin of CH
4
from Merriespruit
is also supported by 16S rRNA gene and by enrichment
cultures which indicate the presence of methanogens
(
Ward et al., 2004
).
In contrast, 16S rRNA gene amplifications for the
Kloof samples (KL739, KL441H2, KL443HWND1,
KL443HWDN) did not yield any evidence of methano-
gens (
Ward et al., 2004; Kieft et al., 2005
). While this
Fig. 1. Plot of
d
13
C versus
d
2
H values for C
1
–C
4
for Kidd Creek (A)
and for 4 new Precambrian Shield sites (B). In the lower panel, the
most
13
C-enriched samples at each site — Driefontein (open squares);
Mponeng (closed circles), Kloof (closed triangles) and CCS (crosses)
all show the characteristic
13
C depletion and
2
H enrichment between
CH
4
and ethane similar to the proposed abiogenic gases at Kidd Creek
(data from (
Sherwood Lollar et al., 2002
) – closed squares– upper
panel). In marked contrast, the pattern for thermogenic gas reservoirs
is a positive correlation of
d
13
C and
d
2
H values for CH
4
and ethane
due to increasing isotopic enrichment in both
13
C and
2
H with
increasing molecular weight for the C
1
–C
4
homologues (open circles
— upper panel). This pattern results from production of the hydro-
carbon gases by thermal cracking of a high molecular weight organic
precursor (
Des Marais et al., 1981; Schoell, 1988
). Error bars on
d
13
C
values are
F
0.5
x
. Error bars on both A and B on
d
2
H values are
F
5
x
and are smaller than the plotted symbols.
B. Sherwood Lollar et al. / Chemical Geology 226 (2006) 328–339
333
may be due to the very low biomass in these samples and
does not entirely rule out a small microbial CH
4
contri-
bution, it is nonetheless consistent with the predominant-
ly abiogenic origin proposed for CH
4
in the Kloof
samples. No microbiological work was carried out at
the other boreholes at Kloof, Copper Cliff South or
Mponeng, but for Driefontein, 16S rRNA gene amplifi-
cation and culture enrichments for methanogens were
attempted for both borehole DR9IPC (results above) and
for one of the proposed abiogenic end-members, bore-
hole DR938H3. While enrichments were unsuccessful,
PCR-amplified 16S rRNA gene indicated a bacterium
(closely related to the sulfate-reducing
Desulfotomacu-
lum
species) and three archaeal taxa within the family
Methanobacteriaceae
. The low biomass in this sample
suggests that concentrations of CH
4
in the 100 uM range
derived from microbial methanogenesis would be insuf-
ficient to isotopically overprint the
N
17 mM abiogenic
CH
4
signature however (
Moser et al., in press
).
The possibility of mixing between microbial hydro-
carbons (characterized by
13
C-depleted and
2
H-enriched
CH
4
values) and abiogenic end-members can be further
evaluated using the traditional scheme shown in
Fig. 3
,
adapted from
Hunt (1996)
. In this plot, microbial hydro-
carbons fall in the upper left corner based on their
13
C-
depleted CH
4
values and CH
4
/ C
2
+
ratios
N
1000 (
Hunt,
1996
). We have modified the scheme to evaluate abio-
genic-microbial mixing, using the abiogenic end-mem-
bers for each site suggested by
Fig. 1
B. For the microbial
end-member, while microbial CH
4
has been identified at
Kidd Creek and Copper Cliff South mines based on
microbial cultures (
Doig, 1994
), no information on the
isotopic composition of that CH
4
exists to date. Whereas
a microbial end-member has been identified at Driefon-
tein (DR9IPC) based on both isotopic evidence and gene
cloning (
Table 3
), we do not have the same information
for Kloof. The same dolomitic Transvaal Supergroup
overlies Kloof mine however and as at Driefontein,
fractures intersecting both the Ventersdorp and overlying
Transvaal provide a conduit for mixing of fluids and
gases between these two formations. For all samples
then, as an initial test of the model, we used a
d
13
C
value of
55
x
for the microbial end-member based
on the carbon isotope value of DR9IPC (
Fig. 3
). By
this approach, data from Kloof are consistent with mix-
ing of the most
13
C-enriched end-member (KL739) with
up to 43% of a microbial hydrocarbon end-member with
a
d
13
C
CH4
value of
55
x
(
Fig. 3
). All estimates are
insensitive to whether the microbial end-member has
CH
4
/ C
2
+
ratios of 10
3
–10
4
or even higher. For Driefon-
tein, DR938H1 is consistent with mixing with up to 84%
microbial CH
4
(based on mixing with DR548), while
mixing between DR9IPC and DR938H3 defines a sep-
arate trend line, as in
Fig. 2
. Given that the gases occur in
hydrogeologically isolated fractured rock it is not sur-
prising that some sites exhibit a range of end-members
and different possible mixing lines, as fracture controlled
systems are rarely homogeneous. In fact, the mixing
Fig. 2. Plot of
d
13
C versus
d
2
H values for CH
4
for sites from this study compared to the conventional fields for microbial and thermogenic CH
4
after
Schoell (1988)
. Except for the two Mponeng samples (see text), samples fall along linear trends from the most
13
C-enriched end-members to more
13
C-depleted and
2
H-enriched end-members. Symbols are as in
Fig. 1
. DR938H3 and DR938H3
V
are samples taken from the same borehole
8 months apart. Unfortunately such temporal sampling was not possible for the other holes, due to the common practice of sealing them immediately
after completion of drilling. Error bars are
F
0.5
x
on
d
13
C values and
F
5
x
on
d
2
H values and are smaller than the plotted symbols. Data from
Masimong (MM) and Merriespruit (MR) are from
Ward et al. (2004)
for comparison. Data from Kidd Creek are from
Sherwood Lollar et al. (2002)
.
B. Sherwood Lollar et al. / Chemical Geology 226 (2006) 328–339
334
lines could be recalculated for a microbial end-member
with a
d
13
C
CH4
value close to MM5 (
60
x
) (
Fig. 4
)
rather than close to DR9IPC (
55
x
), and the data are
still consistent with this simple two-component mixing
between microbial and abiogenic gas, although the esti-
mates of the fraction of microbial mixing vary some-
what. The key point of
Figs. 3 and 4
is not necessarily to
constrain a specific microbial
d
13
C value for each site,
but to demonstrate that the isotopic and compositional
variation in samples observed supports a model of mix-
ing between the
13
C-enriched end-members in
Fig. 1
B,
and microbial end-members with a range of
d
13
C values
typical of CH
4
produced by methanogens. If a similar
approach is taken for the Canadian Shield sites, KC7792
can be explained by a mixture of the most
13
C-enriched
end-member at Kidd Creek (KC8558), with up to 17.5%
microbial CH
4
(
Fig. 3
). The CCS data fit somewhat less
well. CCS4546 is the only sample with abiogenic char-
acteristics at this site (
Fig. 1
B) and is the most
13
C-
enriched and
2
H-depleted value at the site (
Fig. 2
).
Hence, whereas the CH
4
/ C
2
+
ratio for CCS4546 in
Fig.
3
is slightly elevated (8.6 versus approximately 6 for the
other samples), a mixing line through the remainder of
the data to a
13
C-enriched end-member with the
d
13
C
CH4
value of CCS4546 and a CH
4
/ C
2
+
ratio of 6 indicates that
a mixture of up to 23% microbial CH
4
can account for the
rest of the data at that site.
The most
13
C-enriched end-members at each site are
too C
2
+
-rich to be microbial in origin (
Fig. 3
), and
likewise too
2
H-depleted in CH
4
to be consistent with
a thermogenic origin (
Fig. 2
). Analogous to the Kidd
Creek gases, the carbon and hydrogen isotopic patterns
of these gases (
Fig. 1
B) suggest an abiogenic origin.
The fact that the data from these sites in both Canada
and South Africa fall on mixing lines in both
Figs. 2
and 3
provides excellent support for interpretation of
these data as two-component mixing between microbial
Fig. 4. Plot of
d
13
C values for CH
4
versus CH
4
/ C
2
+
ratios adapted
from
Hunt (1996)
. In this plot, mixing lines are calculated as in
Fig. 3
but for mixing between the most
13
C-enriched CH
4
end-member at
each site, and a
13
C-depleted CH
4
rich microbial end-member with a
value of
60
x
(see text).
Fig. 3. Plot of
d
13
C values for CH
4
versus CH
4
/ C
2
+
ratios adapted
from
Hunt (1996)
. Data fall along mixing lines between the most
13
C-
enriched CH
4
abiogenic end-member at each site as suggested by
Fig.
1
B, and a
13
C-depleted CH
4
-rich microbial end-member, with a value
of
55
x
(see text). Mixing lines are calculated based on the
equations:
d
13
C
¼
x
ð
d
13
C
microbial
Þ þ ð
1
x
Þð
d
13
C
abiogenic
Þ
;
where
x
is the percent of the microbial end-member; and,
CH
4
=
C
2
þ ¼ ð
x
ð
I
microbial
Þ þ ð
1
x
Þð
I
abiogenic
ÞÞ
1
;
where
I
= C
2
+ / CH
4
, the inverse of CH
4
/ C
2
+
. The estimated percent
microbial component for each sample (see text) is calculated by pro-
jecting its
d
13
C value onto the mixing lines on
Fig. 3
. As noted in the
text, the estimates of % microbial gas are relatively insensitive to CH
4
/
C
2
+
ratios but are dependent on the selected
d
13
C values for the two end-
members, and hence provide only a conservative estimate of the
relative fraction of microbial gas. In particular, the mixing lines as
depicted assume that the most
13
C-enriched end-member at each site is
entirely free of a microbial contribution. If the most
13
C-enriched
samples are already themselves the product of some mixing with
microbial CH
4
with an even more
13
C-enriched end-member, then
the estimates of the fraction of microbial contribution would be even
larger. Symbols are as in
Fig. 1
. MM5 and MR1 (circles) are microbial
CH
4
end-members previously identified for the Witwatersrand Basin
based on CH
4
isotopic values, 16S rRNA gene and enrichment cultures
(
Ward et al., 2004
). Unfortunately air contamination of sample DR9IPC
means that C
2
+
values are diluted to below detection limit, so a CH
4
/ C
2
+
ratio cannot be calculated for this sample. Based on the similarity in
isotopic composition of this sample to MR1 and MM5, it has been
plotted (in brackets) close to those samples for comparison purposes.
Error bars are
F
0.5
x
on
d
13
C values and
F
7
x
on CH
4
/ C
2
+
ratios and
are smaller than the plotted symbols.
B. Sherwood Lollar et al. / Chemical Geology 226 (2006) 328–339
335
and abiogenic end-members. Alternatively the presence
of a thermogenic component at the sites in this study
cannot be completely ruled out. Indeed,
Ward et al.
(2004)
showed that mixing between microbial and
thermogenic gases might account for gases in boreholes
at Evander mine located in the quartzite, shale and
minor volcanics of the Witwatersrand Supergroup sedi-
ments. Samples from South Africa in the present study
however are predominantly from boreholes in a differ-
ent geologic unit, the basaltic lavas of the Ventersdorp
Supergroup. Furthermore, all the samples at Evander
fall close to the conventional microbial and thermo-
genic isotope fields in
Fig. 2
(
Ward et al., 2004
), unlike
the
13
C-enriched and
2
H-depleted end-members at the
sites in the present study. Hence we feel the best
explanation for the data in the present study is indeed
two-component mixing between
13
C-enriched abiogen-
ic and
13
C-depleted microbial end-members. The fact
that data from this study fit a simple two-component
mixing model between these microbial and abiogenic
end-members suggests that if a thermogenic component
is present, it is small and does not control the isotopic
and compositional variations in a major way.
4.3. H
2
-based autotrophy linked to methanogenesis
This approach to resolving mixing between abiogen-
ic and microbial CH
4
in the crust has important impli-
cations for evolving research into the Earth’s deep
biosphere, specifically the identification of potential
substrates supporting microbial communities in the
deep subsurface. H
2
-based subsurface microbial com-
munities associated with autotrophic methanogenesis
have been proposed in the Columbia River basalt aqui-
fer (
Anderson et al., 1998; Stevens and McKinley,
1995
), Lidy Hot springs (
Chapelle et al., 2002
); and
in carbonate-rich serpentinized peridotites at an off-axis
hydrothermal vent field near the mid-Atlantic ridge
(
Kelley et al., 2001; 2005
). Interest in such systems is
high since they may be modern terrestrial analogues for
subsurface microbial ecosystems under reducing condi-
tions on the early Earth (
Shock and Schulte, 1998
) or
on other planets or Jovian satellites (
Chapelle et al.,
2002; McCollom, 1999
). Several of the boreholes sam-
pled in this study have high concentrations of H
2
gas as
well as the CH
4
and higher hydrocarbons already dis-
cussed. H
2
concentrations in samples from Mponeng,
Driefontein and Kloof are as high as 11.5% (
Table 1
).
H
2
levels in samples from CCS, Kidd Creek and other
sites in the Precambrian Shield rocks of Canada have
been reported in concentrations as high as 1–58%
(
Table 1
; (
Sherwood Lollar et al., 1993b
)). Over several
years, re-sampling of boreholes in Canada has shown
that the high H
2
levels are not simply an artefact of
drilling (
Bjerg et al., 1997
) but a persistent natural
phenomenon in this geologic environment (
Sherwood
et al., 1988; Sherwood Lollar et al., 1993b, 2002
). Like
the hydrocarbon gases, the high quantities of H
2
are
likely the product of abiogenic water–rock interactions
in these geologically isolated, high rock to water ratio
environments. Specific H
2
-generating reactions may
vary from one geological environment to another. At
sites such as Kidd Creek where significant sections of
ultramafic rock occur, H
2
may be the product of ser-
pentinization over geologically long time scales analo-
gous to systems described elsewhere (
Abrajano et al.,
1988; Charlou and Donval, 1993; Kelley et al., 2001
).
In the absence of significant ultramafics, such as at the
Witwatersrand Basin sites, radiolytic decomposition of
water in the uranium-rich Witwatersrand Formation has
been shown to produce H
2
at a sufficient rate to account
for the elevated H
2
concentrations observed in these
isolated fracture fluids (
Lin et al., 2005
). H
2
as an
energy source for autotrophic microbial ecosystems
on both the Earth (
Anderson et al., 1998; Stevens and
McKinley, 1995
) and Mars (
Boston et al., 1992
) has
been a topic of considerable debate in the literature.
Nonetheless, the number of studies that have been able
to convincingly demonstrate coupled oxidation of H
2
and reduction of CO
2
to produce CH
4
via the equation:
4H
2
þ
CO
2
Y
CH
4
þ
2H
2
O
ð
1
Þ
as the basis for a microbial food chain in the deep
terrestrial subsurface has been limited (
Chapelle et al.,
2002
).
The approach for identifying abiogenic and microbial
CH
4
end-members presented in this paper, and the large
database on deep subsurface gases developed by our
research in Canada and South Africa provides an oppor-
tunity to test this proposed reaction on a large scale.
Chemolithoautotrophs growing on H
2
can potentially
reduce a variety of different oxidants including any
available oxygen, sulfur and nitrogen oxidants, metals
and metalloids, as well as CO
2
. If H
2
autotrophy linked to
CO
2
reduction and methanogenesis is an important pro-
cess, there may be a correlation between H
2
utilization
and production of an isotopically
13
C-depleted microbial
CH
4
.
Fig. 5
is a compilation of all samples for Canada
and South Africa measured by our group to date (
n
= 65),
incorporating data from almost two dozen sites. A rela-
tionship exists between H
2
concentrations and the isoto-
pically distinct microbial and abiogenic hydrocarbon gas
end-members identified in this study. With few excep-
B. Sherwood Lollar et al. / Chemical Geology 226 (2006) 328–339
336
tions, high H
2
concentrations (
N
1%) are associated with
only the most
13
C-enriched CH
4
samples (
d
13
C values
between
45 and
25
x
). In contrast, samples with
13
C-depleted CH
4
values (indicative of a significant
methanogenic component) correlate with H
2
values in
the range of 0.1% to 1%, to below detection limit
(
b
0.01%). The observed pattern of H
2
concentrations
and CH
4
isotopic signatures may be the geochemical
fingerprint of H
2
autotrophy linked to methanogenesis
via Eq. (1). This evidence supports the interpretation of
the hydrocarbon isotopic data as controlled by two-
component mixing between
13
C-enriched CH
4
end-
members and
13
C-depleted microbial end-members
and may further suggest that methanogenesis is sup-
ported by H
2
utilization. A scenario could be envisaged
where H
2
–CH
4
rich abiogenic gases are mobilized
when fractures open due to tectonic activity or due to
more recent disturbances due to mining activity and
support a redox gradient-driven ecosystem in the Pre-
cambrian Shield deep subsurface where rapid utiliza-
tion of H
2
by microbial communities quickly depletes
H
2
and produces a
13
C-depleted CH
4
signature charac-
teristic of methanogenesis that mixes with and partially
overprints the pre-existing
13
C-enriched abiogenic CH
4
signature.
5. Conclusions
This study suggests that the abiogenic hydrocarbon
gases first identified at Kidd Creek in fact exist at a
variety of Precambrian Shield sites worldwide. Using
the mixing models outlined here, the relative contribu-
tion of abiogenic versus microbial end-members can be
estimated. Verification of an abiogenic component first
requires identification of the end-member based on the
d
13
C and
d
2
H model illustrated in
Fig. 1
, followed by
evaluation of the extent of mixing with other more
conventional hydrocarbon sources via mixing schemes
such as in
Figs. 2 and 3
. This combined approach has
important implications for evaluation of potential abio-
genic hydrocarbon reservoirs in a wide range of geo-
logic settings, including the longstanding controversy
concerning the possible contribution of abiogenic gases
to economic petroleum hydrocarbon reservoirs (
Gold,
1979; Kenney et al., 2002
). The association of high
concentrations of H
2
with
13
C-enriched CH
4
end-mem-
bers, and the H
2
depletion in
13
C-depleted methano-
genic end-members further suggests the possibility that
abiogenic gases may support H
2
autotrophy linked to
methanogenesis in the deep subsurface.
Acknowledgements
This study was supported in part by grants from the
Natural Sciences and Engineering Research Council of
Canada and Discovery Grants Program, and Canadian
Space Agency to BSL and by the National Science
Foundation Life in Extreme Environments Program
(EAR-9714214) grant to TCO and NASA Astrobiology
Institute grant to the IPTAI team. We thank H. Li for
compositional and isotopic analyses. Special thanks are
due to the geologists and staff of the following mines for
providing geological information and invaluable assis-
tance with underground field work: Kidd Creek Mine,
Timmins, Ontario (D. Duff, R. Cook, P. Olsen); Copper
Cliff South Mine, Sudbury, Ontario (T. Little); and in
South Africa, Driefontein (D. Nel, B. Kotze, M. deKo-
ker), Mponeng (D. Kirshaw, H. Cole, G. Gilchrest) and
Kloof (A. Van Heerden, D. Steinkamp, T. Hewitt, G.
Buxton) Mines. Special thanks are owed to Rob Wilson
of SRK-Turgis for logistical support.
[SG]
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Fig. 5. Histogram of H
2
concentrations versus
d
13
C
CH
4
values for
samples (
n
= 65) from this study as well as for previously reported
data from Precambrian Shield sites in South Africa (
Ward et al., 2004
)
and Canada (
Montgomery, 1994; Sherwood Lollar et al., 1993a,b,
2002
). With few exceptions, high H
2
concentrations (
N
1%) (
n
= 31)
are associated with the most
13
C-enriched CH
4
end-members
(
d
13
C
CH
4
values between
45
x
and
25
x
). In contrast, samples
with
13
C-depleted CH
4
values (indicative of a significant methano-
genic component) have H
2
values between 0.1–1% (
n
= 7), to below
detection limit (
b
0.01%) (
n
= 27).
B. Sherwood Lollar et al. / Chemical Geology 226 (2006) 328–339
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