Applied Geochemistry 143 (2022) 105373

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Applied Geochemistry
journal homepage: www.elsevier.com/locate/apgeochem

Microbial vs abiotic origin of methane in continental serpentinized

ultramafic rocks: A critical review and the need of a holistic approach
Giuseppe Etiope a, b, *, Christopher Oze c
a
Istituto Nazionale di Geofisica e Vulcanologia, Sezione Roma 2, Rome, Italy
b
Faculty of Environmental Science and Engineering, Babes-Bolyai University, Cluj-Napoca, Romania
c
Occidental College, Los Angeles, United States

A R T I C L E I N F O A B S T R A C T

Editorial handling by Dr. Xianjun Xie Continental ultramafic rock systems, through the process of serpentinization, provide chemical and biochemical
pathways that lead to the production of methane. The extent to which rock-water-gas reactions and organisms
Keywords: supply methane in these systems is a matter of considerable discussion and debate. Deciphering the interplay of
Abiotic methane abiotic and microbial methane observed at the surface requires several lines of reasoning as well as a variety of
CO2 hydrogenation
analyses. Despite using multiple models and interpretative tools, conclusions for the origin of methane at a
Fluid inclusions
particular site may vary or diverge from regional or global observations. Here, we critically address how possible
Methanogens
Serpentinization conclusions of microbial versus abiotic methane in continental serpentinization systems may be interpreted and
Peridotite reinterpreted. We review fundamental concepts, advantages and limits, for three major methane origin models:
Hyperalkaline water (a) abiotic CO2 hydrogenation supplying gas reservoirs, (b) derivation from fluid inclusions in olivine-rich rocks,
Ophiolite and (c) microbialgenesis in aquifers. We use the case of methane in the Samail ophiolite of Oman as an
emblematic example of multiple interpretations; we identify ambiguous information offered by methane clum­
ped isotopes and molecular gas compositions (e.g., the meaning of gaseous hydrocarbons heavier than methane),
and suggest key tools, such as radiocarbon (14C) in methane, which may solve interpretative issues. The major
constraint in any model of methane origin is the capability to sustain continuous gas flows, in terms of methane
emission intensity, longevity and spatial extension, such as in natural gas sedimentary systems. Overall, this
review suggests that any site interpretation can benefit from a holistic approach, integrating geochemical,
geological and biological data with gas flow dynamics, as well as including regional and global contextualization.

1. Introduction methane-bearing peridotite systems are the subject of a wide variety of

Methane (CH4), often associated with heavier hydrocarbons (ethane,
C2H6; propane, C3H8), has been reported in aquifers, springs and gas
vents in continental serpentinized ultramafic rock systems (ophiolites,
peridotite massifs and intrusions) worldwide (an updated list of inves­
tigated sites in 20 countries is reported in Table S1). Ultramafic rocks are
devoid of ancient organic matter, thus, methane is generated by pro­
cesses that are different than those producing traditional natural gas in
sedimentary and petroliferous rocks. In sedimentary rocks methane is
derived from ancient life forms, i.e., microbial and thermal decompo­
sition of organic matter. In ultramafic rock systems methane can be a
product of chemical reactions and an energy source for life. In active
serpentinization systems, methane can also be derived from living
chemosynthetic microbial communities (methanogens). Hence,
studies in planetary geology, subsurface microbiology and astrobiology
(e.g., Oze and Sharma, 2005; Schrenk et al., 2013; Etiope and Sherwood
Lollar, 2013 and references therein). The exact origin of such methane
is, however, the subject of current discussion and debate, and three main
alternative or complementary theories have been proposed by different
research groups (Fig. 1): (a) Abiotic methane from low-medium tem­
perature (<150 ◦ C), Fischer-Tropsch Type (FTT) reactions, specifically
CO2 hydrogenation (or Sabatier reaction) in fractures and subsequent
gas migration and accumulation in reservoirs. This model includes the
hypothesis of chromitites as source rocks (Etiope et al., 2018; Etiope and
Whiticar, 2019) and the importance of metals for catalysis. Hereafter,
the term “CO2 hydrogenation-reservoir model” is used.
(b) Abiotic methane from fluid inclusions in olivine-rich rocks,
originated from higher temperatures FTT synthesis after magmatic fluid

* Corresponding author. Istituto Nazionale di Geofisica e Vulcanologia, Sezione Roma 2, Rome, Italy.
E-mail address: giuseppe.etiope@ingv.it (G. Etiope).

https://doi.org/10.1016/j.apgeochem.2022.105373
Received 9 February 2022; Received in revised form 31 May 2022; Accepted 10 June 2022
Available online 15 June 2022
0883-2927/© 2022 Elsevier Ltd. All rights reserved.
G. Etiope and C. Oze
cooling (Klein et al., 2019; Grozeva et al., 2020).
(c) Microbial origin in aquifers, through the action of hydro­
genotrophic (using CO2), formatotrophic (using formate), and/or ace­
totrophic (using acetate) methanogens (e.g., Miller et al., 2018; Nothaft
et al., 2021).
Ultramafic rock systems can also receive variable inputs of thermo­
genic gas from underlying or adjacent sedimentary rocks. This compo­
nent, however, has generally been considered accessory (e.g., Etiope
et al., 2011a; Szponar et al., 2013). Of course, abiotic methane can also
be produced by other mechanisms, as reviewed in Etiope and Sherwood
Lollar (2013), which refer to temperature and pressure conditions that
are much higher than those of continental ultramafic rock systems.
These include the interaction between olivine hydration and dissolved C
in hydrothermal systems (e.g., see a review of the mechanisms in
McCollom, 2016, and a review of the hydrothermal FTT laboratory ex­
periments in Huang et al., 2021), or the reaction beween H2 and
elemental C (graphite; e.g., Deville and Prinzhofer, 2016), which is
theoretically applicable to deep, high T and P systems (French, 1966),
such as subducted slabs (Brovarone et al., 2017). Continental ultramafic
rocks, especially those in ophiolites (with a few exceptions of ophiolites
in high heat flow regions), do not refer to present-day high temperature
hydrothermal systems, as indicated by the generally low geothermal
gradients, temperatures in deep boreholes crossing the whole ophiolite
sequence (e.g., Demirel and Günay, 2000) and modern serpentinization
temperatures (e.g., Ellison et al., 2021). High temperature hydrothermal
processes can be invoked for methane formed within the
mafic-ultramafic rocks before their emplacement on continents, and
preserved in inclusions, as considered in the fluid inclusion model
mentioned above. Methane of pure magmatic origin is also excluded by
multiple lines of evidence, including the relatively low temperatures of
methane formation revealed by methane clumped analyses and other
geo-thermometers (e.g., Etiope and Sherwood Lollar, 2013; Wang et al.,
2015; Blank et al., 2017; Young et al., 2017; Suda et al., 2022).
Here, advantages and limits of the several models and interpretative
tools are examined. A specific discussion develops around the
emblematic case of the gas observed in the Samail ophiolite (Oman) by
several authors who suggested different interpretations (e.g., Neal and
Stanger, 1983; Fritz et al., 1992; Vacquand et al., 2018; Nothaft et al.,
2021). Reflections presented here focus on: (a) the importance of
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Applied Geochemistry 143 (2022) 105373
Fig. 1. A scheme summarizing the three main models
of methane origin in continental serpentinized ultra­
mafic rock systems. Each model must be capable of
generating enough gas amounts and pressures to
sustain the continuous and long-lasting emissions
observed at the surface, including bubble plumes in
springs or surface pools (a), methane in solution
transported by groundwater (b), and dry gas seeps
(c). Photos by G. Etiope: (a) bubbling in one of the
several hyperalkaline springs in the Ronda ophiolite;
(b) CH4-enriched water pumped in the Terme di
Genova spa; (c) flames from the large Chimaera
burning seep in Turkey. Additional photos are re­
ported in Fig. S4.
radiocarbon (14C) CH4 analyses, only occasionally performed in ser­
pentinized ultramafic rock environments; (b) the attribution of a mi­
crobial origin by CH4 clumped isotopes; (c) the interpretation of ethane
and propane data; (d) gas flow dynamics constraints, whereby, any
methane origin, to be acceptable, shall be capable of generating,
continuously and over long-time scales, enough gas to produce those
pressure gradients and gas flows compatible with the relevant occur­
rences and emissions observed at the surface in many places. We show
that a given combination of geochemical data can be explained by
multiple hypotheses, but critical and dynamic relationships that may
solve interpretative problems are identified. Points (a) to (d) reveal the
need of a holistic interpretative approach, capable of incorporating
multiple factors and processes connected to what is specifically observed
in a given site, but that operate on a wider spatial scale, both regional
and global.
2. Three models of methane origin in continental serpentinized
ultramafic rocks
2.1. Abiotic origin related to moderate temperature gas-phase CO2
hydrogenation and accumulation in reservoirs
The pioneering studies of Abrajano et al. (1990), Lyon et al. (1990)
and Fritz et al. (1992) on gas seeps in the Philippines, New Zealand and
Oman ophiolites, and more recent research in Europe, North America,
Japan, New Caledonia and again Oman (e.g., Etiope et al. 2011a; 2013a,
2013b, 2016, 2017; Szponar et al., 2013; Suda et al., 2017; D’Alessandro
et al., 2018; Vacquand et al., 2018), suggest a dominant abiotic origin of
methane (i.e., the gas is not the direct result of microbes or remineral­
ization of organic matter). Variable, but minor biotic (thermogenic or
microbial) components can be present. The abiotic origin refers mainly
to FTT synthesis, mostly low temperature CO2 hydrogenation or Sabatier
reaction (CO2 + 4H2 = CH4 + 2H2O) occurring in fractured rocks.
Although some works consider FTT synthesis inefficient (e.g., McCol­
lom, 2013; Barbier et al., 2020), this conclusion should be restricted to
aqueous CO2 hydrogenation. It is well known that the Sabatier reaction
involves heterogeneous catalysis and operates only in dry, gas-phase
conditions, while aqueous FTT synthesis can proceed only through ho­
mogeneous catalysis, with dissolved catalysts and synthetic ligands
G. Etiope and C. Oze
(Etiope and Whiticar, 2019 and references therein). Laboratory experi­
ments using or promoting gas-phase CO2 hydrogenation (strictly
following Sabatier’s rules) obtained substantial and unambiguous
amounts of methane over a wide range of temperatures (e.g., Etiope and
Ionescu, 2015; Barbier et al., 2020 and references therein). A more
detailed discussion on FTT reactions (with a distinction of CO-based,
Fischer-Tropsch reaction in sensu strictu) and their occurrence in natu­
ral geological systems (gas phase and aqueous solutions) is in Etiope and
Whiticar (2019). The interpretation of this origin was typically based on
multiple lines of evidence, primarily focusing on stable C and H isotopes
composition of CH4 (δ13C and δ2H; Fig. 2), combined with the molecular
gas composition, including H2 and its isotopic composition, preliminary
clumped-isotope analyses (either from surface dry gas seeps or gas dis­
solved in groundwater), the geological-mineralogical context and other
geochemical proxies when possible (Etiope and Sherwood Lollar, 2013;
Etiope and Whiticar, 2019 and references therein). In particular, a
Fig. 3. Δ13CH3D vs Δ12CH2D2 plot. Continental ultramafic rock systems and
Precambrian shield data are from Young et al. (2017), Blank et al. (2017) and
Nothaft et al. (2021). Vertical grey dashed lines are continental ultramafic
systems where only Δ13CH3D was measured (data from Wang et al., 2015;
Woycheese et al., 2017; Cumming et al., 2019; Suda et al., 2022). Submarine
hydrothermal fields data are from Labidi et al. (2020). Laboratory abiotic gas
data are from Young et al. (2017) and Zhang et al. (2021). Blue dashed line
encompasses microbial data (microbialgenesis zone) from Young (2020). (For
interpretation of the references to colour in this figure legend, the reader is
referred to the Web version of this article.)
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Applied Geochemistry 143 (2022) 105373
Fig. 2. Stable carbon and hydrogen isotope ratios of
methane in continental ultramafic rock systems
(ophiolites and peridotite massifs). Redrawn from
Etiope and Whiticar (2019; and references therein;
see also references in Table S1), with the addition of a
burning CH4–H2 rich gas seep in Indonesia (Tanjung
Api; East Sulawesi Ophiolite; Subroto et al., 2004),
the Oman data from Nothaft et al. (2021; only sam­
ples analysed for paired clumped-isotopes are added)
and Precambrian shield data considered dominantly
abiotic (grey crosses; Sherwood Lollar et al., 2006).
Small grey dots refer to biotic (thermogenic and mi­
crobial) gas from sedimentary and petroleum systems
(dataset from Milkov and Etiope, 2018). A similar
diagram including geothermal-volcanic gas and gas in
igneous inclusions is reported in Etiope and Sherwood
Lollar (2013).
proportion of methane was proposed to originate in chromitites as they
showed, among the whole ophiolite sequences in Greece and Brazil, to
be the only rocks with relevant concentrations of CH4, H2 and potential
metal Sabatier catalysts (Etiope et al., 2018; De Melo Portella et al.,
2019). Methane observed in the chromitite microfractures (not in in­
clusions) has the same isotopic composition of surface seeps (Etiope
et al., 2018). It is then hypothesized that the gas progressively migrates
and accumulates in permeable reservoir rocks, in a
source-accumulation-seepage system conceptually similar to natural gas
systems in sedimentary basins (Etiope and Whiticar, 2019). This model
is mainly limited by the poorly known effectiveness and abundance of
minerals that can act as catalysts (e.g., chromium, ruthenium, iron
minerals) that can support FTT reactions over long-time scales at mod­
erate temperature. Although Sabatier reaction based either on artifi­
cially prepared Ru-catalysts or natural chromitite samples has been
demonstrated (Etiope and Ionescu, 2015; Ueda et al., 2021), studies
addressing how metals in natural rocks (generally as oxides or sulfides)
can work as Sabatier catalysts are lacking: can ruthenium minerals (e.g.,
laurite), dispersed at low concentrations (ppb to ppm levels) in the
chromitite, be sufficient to promote methane formation, and what would
be their support (typical of catalysts in Sabatier reactions)? Or is chro­
mium, which is more abundant in ultramafic rocks, a better candidate as
catalyst? If methane is produced at temperatures below 150 ◦ C, as
clumped-isotopes (and geothermal gradients) suggest in several cases,
then the catalyst must be operative at such low temperatures. We know
that ruthenium is effective below 150–200 ◦ C, while other catalysts,
based on Ni and Fe, require temperatures above 200 ◦ C (Etiope and
Whiticar, 2019 and references therein). Chromium is a less studied
catalyst, and we cannot exclude that it can work at low temperatures
over long time scales. It is interesting to note, however, that chromitites
have mineralogical signatures indicative of (a) H2 flow, such as laurite
alteration by desulfurization along microfractures (Garuti and Zaccarini,
1997), and (b) potential CO2-hydrogenation, such as the presence of
amorphous carbonaceous material, typically formed during the reaction
on the catalyst (e.g., Baraj et al., 2016), as observed in some Ru-rich
chromitites in Greece (Economou-Eliopoulos et al., 2019). H2 flow is
also necessary to remove reaction residues and regenerate the catalyst,
thereby, ensuring its activity for prolonged times (e.g., Forzatti and
Lietti, 1999). The available data are, however, scarce; this is a research
area that requires more exploration. CO2 hydrogenation is widely
studied in chemical catalysis, where several metals are tested (e.g., see
review in Wang et al. (2011)), but for improving our understanding of
the Sabatier synthesis in natural rocks, further studies are required.
G. Etiope and C. Oze
Fig. 4. A scheme summarizing pros, cons and unresolved questions for the three main models of methane origin in continental serpentinized ultramafic rock systems.
2.2. Abiotic origin related to fluid inclusions in olivine-rich rocks
Another proposed abiotic origin of methane observed in surface
seeps and springs refers to fluid inclusions in olivine-rich rocks, with
methane originated at higher temperature (>200–300 ◦ C), via FTT
synthesis after magmatic fluids cooling (Klein et al., 2019; Grozeva
et al., 2020). Hydrocarbons leaching from the fluid inclusions would
contribute significantly to surface gas emissions. This model is suggested
by the discovery of CH4-rich fluid inclusions in peridotite and dunite
from ophiolites in Italy, Western United States and the Philippines (Klein
et al., 2019), and by the similitude in gas isotopic composition between
inclusions and surface seeps in the Philippines (Grozeva et al., 2020).
This hypothesis is analogous to the one of McDermott et al. (2015) and
Wang et al. (2018) for methane in submarine hot springs. Its limits are
related to gas flow dynamics constraints, mainly the lack of a model that
can explain how large volumes of inclusion-rich rocks can be involved in
the gas leaching process to assure long-lasting and continuous gas flow
to the surface. Although Klein et al. (2019) hypothesized a process of
fluid inclusion cooling and progressive formation of H2, it is not clear
how fluid inclusions in a given volume of rock, may have, individually
and simultaneously, all the ingredients in the right proportions neces­
sary for CH4 production (H2 ≫ CO2 and catalyst). Available
clumped-isotope analyses of gas discharged at the surface (Fig. 3) also
suggest that methane is formed at temperatures below those typically
considered for gas production in fluid inclusions. It is important to
outline, however, that many CH4-bearing inclusions observed in ultra­
mafic rocks (including chromitites) are secondary and originated by
healing of fractures (Melcher et al., 1997; Van den Kerkhof and Hein,
2001) implying that the gas was initially free along fractures, originated
outside the inclusion, and then trapped within the inclusion. The trap­
ping could take place after the FTT reaction occurred (as in Salvi and
Williams-Jones, 1997), so there would not be a conflict between the CO2
hydrogenation-reservoir model and the inclusions’ model. Klein et al.
(2019) conceived fluid trapping at high temperatures (>400 ◦ C), before
H2 formation.
2.3. The microbial origin
Recent microbiological studies have emphasized the role of microbes
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Applied Geochemistry 143 (2022) 105373
producing CH4 (methanogens), suggesting a frequent, dominant mi­
crobial origin for the gas when it is associated to hyperalkaline (pH > 9)
waters (e.g., Brazelton et al., 2017; Miller et al., 2018; Nothaft et al.,
2021). Methanogens have been detected in some cases (The Cedars in
California, Voltri in Italy, Samail in Oman and Chimaera seep in Turkey;
Kohl et al., 2016; Brazelton et al., 2017; Miller et al., 2018; Zwicker
et al., 2018; Nothaft et al., 2021), but not in others (Cabeco de Vide in
Portugal; Tiago and Veríssimo, 2013; Tablelands in Canada; Morrill
et al., 2014; CROMO in California; Twing et al., 2017; Hakuba-Happo in
Japan; Suda et al., 2022). A dominant microbial component is suggested
by 13C-depleted CH4 values (δ13C < − 50‰) in some hyperalkaline
springs (Bosnia, California, Spain; Fig. 2). New microbiological studies
are proposing that methane can be microbial also when it is
13
C-enriched (even with positive δ13C values), as in Oman, due to
C-limited conditions in peridotites (Nothaft et al., 2021). A more general
microbiological hypothesis, dismissing abiotic theories in general, is
proposed by Xia and Gao (2021), for which the interpretation of an
abiotic origin is acceptable only for methane in fluid inclusions in some
mantle minerals. Besides the observation that methanogens have not
always been detected in ultramafic rock aquifers or springs, a major
limit of the microbial model refers to the fossil, radiocarbon (14C) free
nature of methane analysed so far, while C in the hyperalkaline water,
where methanogens are detected, has measurable 14C quantities; thus,
methane cannot be originated from C in that water. A model explaining
how microbes may use a 14C-free substrate is missing. The study of
endolithic microbial activity within deep mafic-ultramafic rocks may be
a key approach (e.g., Fones et al., 2022), but it should be associated with
the analysis of methane, and its 14C content, occluded in the rocks. In
addition, CH4 production in water appears incompatible with CH4–H2O
disequilibrium generally observed in the hyperalkaline waters (Etiope
and Whiticar, 2019 and references therein). As for the fluid inclusion
hypothesis, a model is necessary to describe how microbes in aquifers
can sustain the pervasive, long-lasting and intense gas flows observed at
the surface in many places (Fig. 1 and Fig. S4). The frequent presence of
heavier hydrocarbons (ethane to butane) indicates that microbialgenesis
must be systematically complemented by other hydrocarbon producing
processes.
Pros, cons and unresolved questions for the three main models of
methane origin are summarised in Fig. 4.
G. Etiope and C. Oze
3. Unravelling biotic vs abiotic CH4 origin by isotopes
13
3.1. The stable “bulk” isotope ratios C/12C and 2H/H of CH4
The analysis of stable C and H isotope composition is an important
step in the study of methane origin, as widely demonstrated on biotic
(microbial and thermogenic) gas in surface biological ecosystems,
sedimentary gas-oil bearing systems (source rocks, reservoirs and seeps)
and geothermal-volcanic manifestations. The fundamental empirical
δ13C–CH4 and δ2H–CH4 genetic diagrams of Schoell (1980) and Whiticar
et al. (1986), and their updated versions of Milkov and Etiope (2018),
are extensively used in gas geochemistry, and provide clues as well as
debates about putative abiotic methane (Etiope and Sherwood Lollar,
2013; Reeves and Fiebig, 2020), including methane from continental
serpentinization systems (Etiope and Whiticar, 2019; Monnin et al.,
2021). An updated version of δ13C–CH4 and δ2H–CH4 data from conti­
nental serpentinization sites is shown in Fig. 2. The variability of
δ13C–CH4 and δ2H–CH4 can be due to genetic (13C and 2H enrichment of
the CH4 precursor, temperature and “maturation” of gas formation) or
postgenetic processes (desorption, oxidation, diffusion, mixing; Fig. S1
in Supplementary Material). It should be noted that this thematic
approach principally relies on empirical fields for δ13C and δ2H, which
are continuously being redefined as new experimental and field data
comes to light. For example, Monnin et al. (2021) indicated that the free
(not dissolved) gas with δ2H–CH4 > − 200‰ is typical of dry gas seeps,
with the exception of Gokdere (a spring in Turkey), while δ2H–CH4 <
− 200‰ is always from springs. We note that Aqua de Ney bubbling
spring also has δ2H–CH4 > − 200‰ and gas dissolved in springs or
aquifers may have δ2H–CH4 > − 200‰ as well (e.g., Tablelands and
CROMO). The variation of δ2H–CH4 seems, therefore, to be only
partially linked to the interaction with the hyperalkaline water.
Notwithstanding the numerous postgenetic factors potentially affecting
the stable C and H isotope composition (Fig. S1), the 13C-enriched
methane of continental ultramafic systems has a relatively homogenous
isotopic genetic field, distinguished from thermogenic and microbial gas
of sedimentary basins. The Samail (Oman) gas, including the samples
with positive δ13C–CH4 values, is however considered dominantly mi­
crobial by Nothaft et al. (2021).
3.2. The CH4 clumped isotopes
The advent of multiply-substituted “clumped” CH4 isotopologues
analyses (13CH3D and 12CH2D2, see review in Young, 2020) has allowed
additional genetic and post-genetic information to elucidate on tradi­
tional “bulk” isotope analyses (δ13C and δ2H), specifically regarding the
temperature of CH4 formation (Stolper et al., 2015; Young, 2020 and
references therein). CH4 clumped isotopes have helped to distinguish
between microbial, thermogenic, and very high temperature abiotic
(magmatic, hydrothermal) origins (e.g., Stolper et al., 2018; Wang et al.,
2018; Giunta et al., 2019; Young, 2020). The assessment of the CH4
formation temperature is possible only when methane isotopologues are
in thermodynamic equilibrium (Young et al., 2017). This generally
happens for methane formed at high temperatures, >150 ◦ C (thermo­
genic and magmatic/hydrothermal gas). Clumped isotope analyses
revealed that microbial gas (from lab cultures and wetlands) is in iso­
topologue disequilibrium, with a specific range of combinations of low
to negative Δ13CH3D and negative Δ12CH2D2 values (Wang et al., 2015;
Young, 2020 also for details on the delta notation in per mil units).
Methane elsewhere with such a range of combinations (expressed as a
genetic zonation in a Δ12CH2D2 - Δ13CH3D diagram; Fig. 3) is, therefore,
considered very likely microbialgenic. Such a conclusion was applied to
the methane sampled in some wells in the Samail ophiolite in Oman
(Nothaft et al., 2021), an area where the gas, sampled in surface seeps
and springs, was formerly considered mainly abiotic (Fritz et al., 1992;
Vacquand et al., 2018), and which will be discussed in more detail in
Section 5.
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Applied Geochemistry 143 (2022) 105373
Beyond Oman, paired (Δ12CH2D2 and Δ13CH3D) clumped isotope
analyses are today available from four sites of serpentinized ultramafic
rocks, in Italy, Portugal, Turkey (Young et al., 2017) and California
(Blank et al., 2017); from five other sites (in California, Canada,
Philippines and Japan) only Δ13CH3D is available, thereby, not allowing
equilibrium or disequilibrium, and therefore the temperature, to be
assessed. The published data are shown in the Δ12CH2D2 vs Δ13CH3D
diagram of Fig. 3.
The data reveal that only two cases, Chimaera and Aqua de Ney,
show isotopologue equilibrium or near-equilibrium (suggesting a for­
mation temperature of around 130 ◦ C and 50 ◦ C, respectively; Young
et al., 2017; Blank et al., 2017), while the other cases fall above or below
the equilibrium line (Fig. 3). The reasons of these deviations from
disequilibrium are elusive. Available data and models suggest that iso­
topologue disequilibrium, beyond being typical of relatively low tem­
perature reactions, can be due to a series of genetic and post-genetic
processes: (a) kinetics, (b) mixing between abiotic endmembers or be­
tween abiotic and biotic (microbial or thermogenic) endmembers, (c)
fractionation of isotopologues by molecular mass, (d) oxidation (e.g.,
Anaerobic Oxidation of Methane, AOM), (e) combinatorial effects,
and/or (f) tunneling (Wang et al., 2016; Young et al., 2017; Taenzer
et al., 2020; Young, 2020; Warr et al., 2021; Fig. S1). In particular, when
a reaction uses hydrogen from different sources, with a different 2H/H
ratio, then a low Δ12CH2D2 results (combinatorial effect; Taenzer et al.,
2020). Combinations of two or more of the processes (a) to (f) can lead to
a wide range of Δ12CH2D2 vs Δ13CH3D values, including those typical of
microbialgenesis. Therefore, it is not possible to define unambiguous
genetic zonations in the Δ12CH2D2 vs Δ13CH3D diagram. It is worth
noting that methane of Cabeço de Vide (Portugal) falls within the
microbialgenic zone, but methanogens were not detected in the water
(Tiago and Veríssimo, 2013). It is also worth noting that when only the
value of Δ13CH3D is measured, the assumption that methane formed at
the relative “apparent” temperature is deceptive: for example, Suda
et al. (2022) developed an abiotic CH4 origin model for the Happo site
(Japan) assuming the apparent temperatures of about 200–300 ◦ C
derivable from Δ13CH3D values of ~2.4 and 1.5. But similar values were
reported for Samail and Acquasanta gas (sites with lower geothermal
gradients than Happo), which are not in isotopologue equilibrium and
the real temperature cannot be defined. The case of the CH4 generated in
laboratory at 70 ◦ C (Young et al., 2017), whose apparent Δ13CH3D
related temperature would be ~150 ◦ C (Fig. 3), is illuminating.
Clumped isotopes for methane of certain abiotic origin, as it is pro­
duced in laboratory, are available from three different experiments, with
low and high temperature FTT synthesis and high temperature silane
(Si5C12H36) decomposition (Young et al., 2017; Zhang et al., 2021). The
high temperature silane-derived gas is in isotopologue equilibrium,
while the low temperature Sabatier reaction gas has a strongly negative
Δ12CH2D2 value (Fig. 3). Interestingly, the 340 ◦ C FTT experiments of
Zhang et al. (2021) showed methane initially with Δ13CH3D values from
+1.3 to − 1.6‰ (anti-clumped) and Δ12CH2D2 = − 45‰, falling within
the microbialgenic zone in the plot of Fig. 3. Both clumped isotope
values gradually increased, as for bond reordering, and finally
approached the thermodynamic equilibrium at 340 ◦ C in about 3 h. This
experiment showed that during FTT synthesis the clumped isotopes can
evolve during the reaction, and that early methane produced and
released from the system (reactants plus catalyst) is not in thermody­
namic (isotopologue) equilibrium. Obviously, there is the problem of
knowing if and how the early clumped-isotope signature, observed
rapidly after the start of the reaction, is observable in natural gas. We
don’t know whether fast evolution laboratory FTT products are appli­
cable to natural FTT processes. The time-scale issue is a general problem
for any attempt to simulate nature with lab experiments. In this respect,
we can consider processes associated with thermogenic methane pro­
duced in and released from shales and coal, and apply them here. There
are three processes inducing isotopic evolution of gas observable from
shales and coal: (a) maturation; (b) desorption; (c) diffusion.
G. Etiope and C. Oze
(a) Methane produced by catagenesis follows a 13C-increase evolu­
tion (maturation), so that the first methane molecules that are
produced (early mature) and liberated out of the shale/coal are
not in thermodynamic equilibrium and are more 13C depleted;
methane produced later, over longer time scales will be more 13C
enriched. The effects of this on clumped-isotope composition are
discussed by Xia and Gao (2019).
(b) Once formed in shales or coal, much of methane is adsorbed on
the surface of minerals and organic matter. When depressuriza­
tion begins (for geological reasons, uplift, changes of lithostatic
pressure, etc.) methane enriched in 12C desorbs more rapidly than
13
C-enriched methane. The residual methane, liberated in later
stages, is more 13C-enriched. This process is also a cause of
isotope trend reversal, because residual methane becomes
enriched in 13C more rapidly than ethane (Milkov et al., 2020).
(c) Free gas in shale/coal migrates towards other rocks by diffusion-
advection (generally driven by decompression), and the diffusive
movement induces isotopic fractionation (i.e., the first methane
diffusing out is more 12C-enriched).
In natural FTT synthesis it is plausible to have similar processes,
whereby, the degree of FTT reaction can be seen as a “maturation” (the
first liberated gas is early mature and not in isotopologue equilibrium);
desorption is actually a step foreseen in FTT reactions (desorption from
the catalyst); what Zhang et al. (2021) observed is likely a first liberation
(desorption) of methane, which is in isotopologue disequilibrium. Zhang
et al. (2021) also found an increase of δ13C over time, similar to the low
temperature Sabatier experiments (Etiope and Ionescu, 2015). Stolper
et al. (2018) mentioned the possibility that desorption from shales could
be expressed as kinetic isotope effects influencing methane
clumped-isotope composition. Diffusion can also induce
clumped-isotope fractionation (Fig. S1).
Basically, we cannot exclude the possibility that, also in natural
geological conditions, early abiotic gas generated in a CO2 hydrogena­
tion reaction may have low clumped-isotope values, and that residual
(late-desorption) and/or mature abiotic gas has higher values. Accord­
ingly, residual and/or mature abiotic gas would be in isotopologue
equilibrium, if formed at relatively high temperature; early abiotic gas
would be always in disequilibrium and could have Δ13CH3D and
Δ12CH2D2 values similar to those of microbial gas.
4. Holistic contextualization of local observations
The interpretation of CH4 origin at a given site cannot be based solely
on what is observed in that site. If a gas seep or gas-rich aquifer is part of
a wider regional gas system and similar systems exist at global scale,
then the whole regional-global system must be considered to understand
the local processes. Geochemical data should include gas species that,
even if they are not directly related to methane origin (e.g., N2, noble
gases), may reveal crustal or mantle degassing processes. Geochemical
data should then be integrated with geological, structural and petro­
graphic factors, e.g., the relationships between gas discharges and faults,
distribution of ultramafic rocks (especially those with metals that can be
potential catalysts of FTT reactions), C bearing rocks and sedimentary
formations. Not least, the interpretation of gas origin should be
compatible with quantitative aspects, such as the gas flow and emission
on the surface. A holistic approach may offer some answers, as discussed
below. This approach is then used to critically review the case of Samail
gas in Oman, as discussed in Section 5.
4.1. Regional and global contextualization of local data
In several cases, the occurrence of gas in a specific site, seep, spring
or aquifer, is the local expression of a wide, regional gas seepage system.
Examples of regional seepage systems in ultramafic rocks, with multiple
gas seeps or springs distributed over areas hundreds to thousands square
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km wide, are in the Voltri ophiolite (Italy), Samail ophiolite (Oman),
Ronda peridotite massif (Spain), Dinaride ophiolite (Bosnia-Herzego­
vina) and Kizildag ophiolite (Estern Turkey; Table S1 and references
therein). These systems often reveal the existence of crustal degassing
processes, which should be considered to interpret local data. A common
(or dominant) origin of methane may exist in all gas occurrences of a
given regional system. Examples of regional contextualizations are in
Etiope et al. (2016), D’Alessandro et al. (2018) and Vacquand et al.
(2018).
For the interpretation of local data, a global perspective on gas in
continental serpentinization systems is also fundamental. An example of
this type of consideration was explored in Monnin et al. (2021), where
they compared molecular and isotopic gas composition from different
continental serpentinization sites. Differences between methane dis­
solved in water and in dry gas seeps and specific relationships between
methane and other gases (H2 and N2) were identified. A gas origin
sequence model was proposed, whereby, H2 is first produced in water by
serpentinization, then it reacts in gas-phase with CO2 (Sabatier reaction)
producing CH4. The methane can subsequently migrate and dissolve in
water where microbial (methanotrophic and methanogenic) activity
takes place. This coincides with the CO2 hydrogenation – reservoir
model of Etiope and Whiticar (2019).
Some holistic questions are suggested here only for reflection pur­
poses. Can the three theories of gas origin (i.e., microbialgenesis, abiotic
from inclusions, abiotic from CO2 hydrogenation-reservoir) be valid
systematically, everywhere in all 20 countries where methane is
observed to stem from ultramafic rocks (Table S1, and the map in Etiope
and Whiticar, 2019), or are they complementary, each of them valid
only in some cases and not in others? For example, can Chimaera gas be
dominantly abiotic, while Oman gas is microbial and Philippines gas is
mainly from inclusions? Should we look for a unique (dominant) gas
origin, common to all cases, or all three origins may occur simulta­
neously, in variable proportions, in all sites, so that the gas is always
mixed? Mixtures of the three origins at the same site are theoretically
possible because all ophiolites or peridotite massifs can have all items
needed for the three origins: present-day serpentinization (= H2 and
energy sources for methanogens), peridotites with fluid inclusions,
metal (catalyst)-rich rocks and fracture networks for migration of gas
(including inputs of CO2 from external sources). Nevertheless, it is
plausible that one genetic process dominates at the global scale: this
could be preliminarily identified by weighing the pros and cons deter­
mined by available data (Fig. 4), and considering one fundamental
factor, often neglected in gas-geochemical studies: how to explain the
intense and long-lasting methane flow observed at the surface. This is
discussed in the next sections.
4.2. The clumped-isotope perspective
If clumped-isotopes are used to unravel the gas origin, we note that
methane in three serpentinization sites, Acquasanta (Italy), Aqua de Ney
(California) and Chimaera (Turkey), do not fall within the micro­
bialgenic zone of Fig. 3, and, therefore, should not be considered
dominantly microbial (unless other processes are invoked to demon­
strate that also microbial gas can have non-microbialgenic clumped-
isotope signatures). So, based on the current clumped isotope interpre­
tation (e.g., Young, 2020; Nothaft et al., 2021), not all sites are domi­
nated by microbial gas. We note also, as mentioned above, that Cabeço
de Vide (Portugal) falls within the microbialgenic zone, but it lacks
methanogens (and has 13C-enriched CH4 and C2+ alkanes; Tiago and
Veríssimo, 2013; Etiope et al., 2013b).
Methane from fluid inclusions (high temperature hydrothermal or
post-magmatic origin) should be in isotopologue equilibrium and indi­
cate a relatively high temperature, like the data from submarine hy­
drothermal fields (Fig. 3). Only two sites, Chimaera and Aqua de Ney,
are in or near isotopologue equilibrium, but at temperatures lower than
150 ◦ C. The Oman and Portugal sites, being below the equilibrium line,
G. Etiope and C. Oze
are not likely dominantly from inclusions. Based on clumped isotopes,
no site gas would dominantly come from inclusions; unless post-genetic
processes modify the clumped-isotope composition.
Methane from CO2 hydrogenation would have a wider clumped-
isotope signature range, depending on the disequilibrium-equilibrium
evolution during the catalyzed reaction, involving desorption and
diffusion fractionations, as discussed above. Accordingly, the clumped-
isotope composition would not be a single diagnostic of abiotic CO2
hydrogenation. The data related to the several continental serpentini­
zation sites are actually sparse in the Δ13CH3D vs Δ12CH2D2 plot (Fig. 3).
4.3. The quantitative factor: gas flow dynamics constraints
A key factor that should not be neglected is the amount of methane
emission at the surface. Many continental serpentinization seeps or
springs release significant amounts of methane, such as the Chimaera
seep in Turkey (several hundreds of tonnes per year; Etiope and Schoell,
2014), the Archani-Ekkara springs in Greece or the Acquasanta seepage
system in Italy (hundreds of kg per year; Etiope et al., 2013a; Etiope and
Whiticar, 2019). Methane fluxes were not measured at the >60
springs/seeps in Oman, but the water flow rates, active bubbling, dry gas
seeps and the wide area of seepage suggest that the overall emission is
relevant. Other sites with significant transport of methane to the surface,
either through multiple springs, bubble plumes or dry gas vents, are
documented in Eastern Turkey (D’Alessandro et al., 2018), Southern
Spain (Etiope et al., 2016), Philippines (Los Fuegos Eternos; Vacquand
et al., 2018) and Indonesia (Tanjung Api “eternal flames”; Subroto et al.,
2004; Fig. S4). Can these continuous and long-lasting emissions be
supported by the three invoked methane sources individually (micro­
bial, abiotic inclusions, CO2 hydrogenation-reservoir model)? Visible
gas emissions, either as bubbling in water or as dry gas seeps, are clearly
advective, driven by pressure gradients. This implies that the gas source
is pressurized, from accumulation, or that the gas generation process
(the “kitchen”) is very prolific, continuous and fast enough to maintain
the surface output. We think this second hypothesis is not realistic, as
gas production rates in geology are typically very slow. The advantage of
having a reservoir, like in the CO2 hydrogenation-reservoir model, is
that the gas production must not necessarily be continuous and intense;
the gas can progressively form over geological time-scale, with rela­
tively low production rates (as estimated in Etiope and Whiticar, 2019),
and gradually migrate towards permeable rocks, exactly as in a petro­
leum system. The microbial and fluid inclusion theories need to be
completed with a satisfactory gas flow model. In particular, the aquifer
microbial theory should explain how methane can accumulate (with
pressure) above the aquifer (Fig. 4). The microbial model shall, then,
explain where (at what depth and in which rock) methanogens operate
and how can they feed, also in the spatial dimension, the multiple gas
discharges with advective flows (via gas accumulations or through fast
gas generation?). The C-feedstock of methanogens should also be
enough to match the final CH4 output. A picture of the carbon cycle,
examining availability of formate, acetate, CO and atmospheric CO2,
should be developed. Doubts exist, in fact, on sufficient availability of
organic acids and CO compared to the amounts of CH4 released (Etiope
and Whiticar, 2019). The fluid inclusion model shall describe how and
why methane is leached from fluid inclusions, the volumes of rocks with
the gas-rich inclusions that should be involved and how, once a given
volume of inclusions is evacuated, new inclusions in other volumes of
rocks (laterally or vertically distributed) are involved, always producing
the relevant and continuous gas flows observed at the surface. Basically,
how would fluid inclusions continuously rupture/leak on such a time
and geographic scale? This fluid dynamics perspective is essential in
order for the multiple gas origin hypotheses to be both geologically and
physically realistic.
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4.4. Fluid inclusions as a source of surface gas seeps
The presence and concentration of methane in fluid inclusions has
created debate surrounding its origin as well as how it may supply a
continual and pressurized gas source observed at the surface in the form
of dry gas seeps and/or associated with springs (Etiope and Whiticar,
2019; Klein et al., 2019). Olivine hydrolysis experiments, direct mea­
surements of methane (and other gases) in olivine fluid inclusions, and
isotope analyses from field locations provide the basis/start to deter­
mine how olivine can directly contribute methane in these systems. The
complexity of this issue can be highlighted by the case history of the San
Carlos olivines (Arizona, USA), which were studied to assess whether
methane in inclusions can represent a background source in laboratory
FTT experiments. In these olivines, McCollom (2016) measured a wide
range of methane concentrations, by a factor of 49. But Pasteris and
Wanamaker (1988) and Smith et al. (2017) did not detect any methane.
The presence of CH4 in microfractures and veins in igneous rocks, rather
than in inclusions, has been observed in several cases (e.g., Potter and
Konnerup Madsen, 2003; Nivin, 2016), including ultramafic rocks
(Etiope et al., 2018). Therefore, modeling how and to what extent ol­
ivines can contribute methane solely from fluid inclusions is difficult,
especially when attempting to scale up.
Expanding on other studies, the abundance of methane determined
by crushing whole rocks (bulk analysis), as performed by Grozeva et al.
(2020), does not represent exclusively gas from inclusions. To be sure
that in crushing analyses gas derives solely from inclusions, it would be
necessary to completely isolate olivine crystals, as done in Salvi and
Williams-Jones (1997). In the context of fluid inclusions, what is true for
one olivine is not the same for olivine sampled from the same location,
let alone olivine from different locations.
There is, then, a debate on whether fluid inclusions can support the
large amount of methane released over 2000 years from the Chimaera
seep in Turkey (Etiope and Whiticar, 2019; Klein et al., 2019); the dis­
cussion was around approximate calculations (Etiope and Whiticar,
2019) based on extreme assumptions and values of parameters
(maximum volume of fluid inclusions in igneous rocks, 0.01% of rock, as
indicated by Roedder (1972); 100% of CH4 in each inclusion; very
conservative amount of methane, 0.5 km3, produced over only 2000
years) considered just to derive the very minimum theoretical volume,
in terms of order of magnitude, of rock containing inclusions. Using a
range of methane partial pressure observed in inclusions, Klein et al.
(2019) derived a range of 1383 to 10 km3 of rock, and considered that
fluid inclusions cannot be excluded as a source of the Chimaera gas.
Details of the calculations and related debate are reported in the Sup­
plementary Material. Whether or not the range 101–103 km3 of rock is
realistic (considering the need to convey the gas from this rock volume
to a focused area on the surface, continuously and for very long time, as
discussed in Section 4.4), we reiterate that the derived numbers refer to
extreme values, not representative of the average rock conditions.
Methane concentration is not 100% in all inclusions, the volume of fluid
inclusions in igneous rock is often <0.01% of rock and the amount of
methane released by the Chimaera seep since its origin (which certainly
predates 2000 years) is much higher than 0.5 km3. Using average values
of the several parameters, volumes of peridotite rocks at least in the
order of 104 km3 of rock can be derived (Supplementary Material, S1).
There is a need for more realistic values and modeling that still needs to
be completed.
5. The case of methane in the Samail ophiolite
An interesting and complex case of multiple interpretations for
methane origin in ultramafic rock settings is the one of the aquifers and
springs in the Samail (or Semail) ophiolite in Oman. This region hosts
numerous sites, probably more than 60, with hyperalkaline springs and
dry gas seeps where methane is documented together with H2 and N2
(Neal and Stanger, 1983; Fritz et al., 1992; Sano et al., 1993; Vacquand
G. Etiope and C. Oze
et al., 2018 and references therein). Vacquand et al. (2018) showed that
a complex gas-water seepage system exists along a belt of >350 km
comprising several massifs of the Samail ophiolite, and that the gas has
variable CH4, H2 and N2 compositions. In all cases, a dominant abiotic
CH4 component was suggested based on multiple gas-geochemical data,
in agreement with earlier studies (Fritz et al., 1992; Sano et al., 1993).
The recent study by Nothaft et al. (2021) focused on fluids intercepted
by a series of wells drilled (max depth of 400 m) in the framework of the
Oman Drlling Project, in the southern Wadi Tayin Massif, near Ibra.
Based on microbiological (16S rRNA Gene Sequencing) and fluid
geochemical analyses including CH4 clumped isotopes, Nothaft et al.
(2021) claimed that methane in the Samail ophiolite is mainly micro­
bial, with a minor abiotic component derived from fluid inclusions in the
peridotite. The microbial origin was suggested on the basis of: (a) the
presence of methanogens in the water intercepted by the investigated
wells (water pumped from depths mostly <100 m), and models pointing
to energetically favourable formatotrophic methanogenesis. Metha­
nogens can produce 13C-enriched CH4 in C-limited conditions (perido­
tite aquifer); (b) low values of CH4 clumped isotopes Δ12CH2D2 and
Δ13CH3D (considered to be characteristic of microbialgenesis, as dis­
cussed above).
The subordinate fluid inclusion origin was suggested by: (c) the
occurrence of 13C-enriched methane, ethane and propane, similar to
those found in seafloor hydrothermal vents where fluid inclusions are
considered a dominant source of alkanes (e.g., Grozeva et al., 2020); (d)
literature reference of some, but not all applicable, CH4-bearing in­
clusions found in the Samail peridotite (Miura et al., 2011). Nothaft et al.
(2021) did not include the abiotic CO2-hydrogenation-reservoir model
(where chromitites can be gas source rocks), claiming that: (e) there is
not enough free H2 or CO2 for FTT catalysis; (f) there are no chromitites
(catalyst-rich rocks) in the area, neither in rocks intercepted by the
investigated wells or in surrounding outcrops. Points (b) to (f) are
examined in detail in the following sections. Local data are contextu­
alized considering the wider Samail gas seepage system and the valuable
addition of measuring the radiocarbon (14C) content of CH4 is discussed.
5.1. Low values of CH4 clumped isotopes Δ12CH2D2 and Δ13CH3D are not
exclusive of microbialgenesis
Clumped isotopes of methane sampled in three wells in the Samail
ophiolite have a Δ13CH3D versus Δ12CH2D2 combination typical of
microbialgenesis (Nothaft et al., 2021). This is a major point used by
Nothaft et al. (2021) to support their microbial origin hypothesis. This
point loses strength when a more rigorous analysis of the numerous
factors influencing the clumped isotopic composition and the recent
laboratory FTT data, discussed above, are considered. Mixing, frac­
tionation of isotopologues by molecular mass, desorption, oxidation,
kinetics and tunneling (Young, 2020), complicate the interpretation of
clumped isotopes and can lead to signatures similar to those of micro­
bialgenesis (Fig. S1). The FTT experiments of Young et al. (2017) and
Zhang et al. (2021), and the case of Cabeço de Vide mentioned above
(where methanogens were not detected), actually showed that abiotic
gas can have Δ13CH3D and Δ12CH2D2 signature similar to microbial gas
(Fig. 3). The Samail ophiolite CH4 requires additional clumped isotope
analyses, especially of samples taken in surface discharges, preferably
the dry gas seeps (as those studied in Vacquand et al., 2018), which are
not considered in Nothaft et al. (2021).
5.2. The fluid inclusion hypothesis
5.2.1. Existence and abundance of fluid inclusions
In the Samail ophiolite, an additional gas origin from fluid inclusions
was hypothesized by Nothaft et al. (2021) to explain the minor presence
of 13C-enriched ethane and propane in the observed wells. Although the
authors write that this “is evidenced by the presence of CH4-bearing fluid
inclusions in the Samail Ophiolite,” their work did not report any analysis
8
Applied Geochemistry 143 (2022) 105373
of fluid inclusions supporting the abundance of alkanes detected in the
wells, but refer to Miura et al. (2011), who reported Raman analysis of
only one harzburgite sample from another massif, about 280 km north of
the area (the Wadi Tayin Massif) studied by Nothaft et al. (2021). Miura
et al. (2011) reported that the inclusions are secondary, referring to gas
trapped in healed fractures, and that most of the inclusions are gas-free.
Using this reference (one sample, with rare CH4-inclusions, far from the
studied area) to support the hypothesis that methane in the Samail
ophiolite comes from inclusions in local peridotites is, at least,
premature.
There are no studies on the methane-inclusion mass balance in pe­
ridotites. The capability of the inclusions to support long-lasting and
large gas emissions on the surface, as observed in some places (e.g., the
Chimaera burning seep in Turkey) is the subject of a specific debate
(Etiope and Whiticar, 2019). A detailed discussion on this aspect is re­
ported in Section 4.4 and Supplementary Material. A critical question,
discussed in Section 4.3, concerns the gas flow mechanics: what is the
mechanism of methane leakage from inclusions that involves progres­
sively different portions of the peridotite massif, after that the inclusions
from a certain volume of rock are evacuated, and assuring continuous
advective gas flows driven by intense pressure gradients? A
geological-fluid dynamic model, including mass-balance, should be
developed to answer the question. For the Samail ophiolite seepage belt
(>350 km long), the continuous release of methane in at least 60
different places (whether from geological or historic times is unknown)
would require an incredibly huge volume of rock with high concentra­
tions of CH4-rich inclusions.
5.2.2. The meaning of the C1/(C2+C3) ratio
The gaseous alkanes heavier than methane (i.e., ethane, propane,
and occasionally butane) have been widely reported in continental
serpentinization seeps and springs. Their presence was documented in
all sites where clumped-isotopes were also measured (Fig. 3). The C1/
(C2+C3) ratio (methane/(ethane + propane), also called “Bernard
ratio”) is frequently used to support the δ13C vs δ2H diagram to better
understand the gas origin. An updated version of the genetic zonations
in the δ13C vs C1/(C2+C3) diagram, based on a wide biotic and abiotic
dataset, is reported in Milkov and Etiope (2018). While this diagram can
be mostly useful to distinguish (with some caution) between microbial
and thermogenic gas, it has no value for gas in igneous rocks,
hydrothermal-magmatic systems and serpentinized ultramafic rock
systems (putative abiotic gas), since this gas has a wide range of
C1/(C2+C3) values (Milkov and Etiope, 2018). In addition, it is known
that the C1/(C2+C3) ratio is an ambiguous genetic proxy because ethane
and propane concentrations can decrease during gas migration (mo­
lecular fractionation), due to differential adsorption and solubility (e.g.,
Deville et al., 2003; Etiope et al., 2009). For example, gas in surface
seeps often has less ethane and propane, relative to methane, than in
reservoirs, which in turn have less ethane-propane compared to the
source rocks (Milkov and Etiope, 2018). It seems that the C1/(C2+C3)
ratio on the surface depends on the intensity, velocity of the gas flow
from the reservoir or source rock (the higher the velocity, the lower the
residence time in the subsurface, the lower the molecular factionation).
The same can happen for abiotic gas. Furthermore, abiotic gas produced
by FTT synthesis can have a wide range of C1/(C2+C3) ratios, since the
amount of several alkanes depends on the degree of methane polymer­
ization, which is function of temperature and time (Sherwood Lollar
et al., 2008). Therefore, the C1/(C2+C3) ratio of abiotic gas does not
have a specific signature.
In the gas observed in the Samail ophiolite well NSHQ14, there are
significant amounts of ethane and propane, and this led Nothaft et al.
(2021) to consider an additional non-microbial gas component. It is
worth noting that Samail gas also has heavier alkanes, butane to hexane
(Neal and Stanger, 1983; Nothaft et al., 2021), which would turn the
attention towards a possible sedimentary, thermogenic origin. Nothaft
et al. (2021) reported a C1/(C2+C3) ratio from 1240 to 881, and claimed
G. Etiope and C. Oze
that these values are not representative of low temperature abiotic
origin, because they are more similar to those in fluid samples and rocks
from other ophiolites and sediment-poor seafloor hydrothermal vents,
and are much higher than those reported for low temperature abiotic gas
observed in Precambrian shields (Kidd Creek, Canada). As explained
above, however, this approach to decipher the gas origin by comparing
C1/(C2+C3) with significantly different abiotic systems, without
considering the context, risks misinterpretation.
In the Table 4 presented in Nothaft et al. (2021), it seems that the
C1/(C2+C3) ratio was calculated using dissolved gas concentrations
values (which depend on the solubility of the gas), which cannot be
compared to the ratios derived from free gas phases, as completed for the
Kidd Creek gas (and as generally done for the Bernard diagram). Un­
fortunately, the original composition of gas extracted from water was
not reported. The fluid inclusion hypothesis shall then be better justified
by explaining how butane to hexane alkanes form in the inclusions.
5.2.3. The stable C isotopic composition of ethane and propane
13
C-enrichment in ethane and propane is generally an indication of
the high maturity of thermogenic gas. It may also be an effect of post-
genetic processes (oxidation, isotopic fractonation by diffusion). The
latter may apply to abiotic gas. For the Samail ophiolite gas, Nothaft
et al. (2021) report a stable C isotopic composition of ethane and pro­
pane of only one sample (NSHQ14 – 2017; δ13C2: +6‰, δ13C3: +3.3‰).
Combined with the non-detection of C2–C3 oxidising bacteria in the
NSHQ14 well water, the 13C-enrichment was considered to reflect the
formation conditions. As the isotopic values are similar to those
observed in fluid inclusions in rocks from the oceanic lithosphere, it was
assumed that the gas cannot be derived from low temperature CO2 hy­
drogenation. This is only one of the several solutions of the problem.
Other processes leading to 13C-enriched C2 and C3 cannot be excluded a
priori:
(a) If alkanes are not produced in the water (as Nothaft et al., 2021
assumed considering the fluid inclusion origin), they could be
oxidised before dissolving into the investigated water, for
example within deeper pore/fracture water.
(b) C2–C3 may be derived from polymerization of 13C-enriched CH4
(Sherwood Lollar et al., 2008). 13C-enrichment in CH4 can be, in
theory, due to methane oxidation or the formation from a
13
C-rich precursor. Such a precursor could be the CO2 derived by
thermal decarbonation (Wycherley et al., 1999) from the
13
C-enriched Permian carbonates, such as the Hawasina,
Sumeini, or Saih Hatat formations occurring below and in lateral
tectonic contact with Samail ophiolite nappe (Hopson et al.,
1981).
(c) 13C-enriched C2–C3 (as well as CH4) may represent residual gas
isotopically fractionated due to late desorption and diffusion in
low permeability rocks (as discussed above with reference to the
evolution of clumped-isotope composition). 12C-enriched gas
desorbs and diffuses faster than 13C-enriched gas; residual gas is
then 13C-enriched. This phenomenon is typically observed in
sedimentary gas source rocks, but it can occur in any low
permeability system.
Accordingly, 13C-enriched ethane and propane in the Samail ophio­
lite NSHQ14 well may not necessarily come from fluid inclusions, whose
abundance in the area investigated was not assessed, as discussed in
Section 5.2.1.
5.3. Availability of chromitites and free reactants for catalyzed CO2
hydrogenation
CO2 hydrogenation needs metal catalysts, therefore, methane should
be produced in a metal-rich rock. In ophiolites, potential metal catalysts
are particularly abundant in chromitites, typically occurring in the
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tectonite basal ultramafic member. Etiope et al. (2018) found that
among all rocks composing four different ophiolites in Greece, only
chromitites host relevant amounts of methane, hydrogen and C2+
heavier alkanes. A similar finding was reported by De Melo Portella et al.
(2019) for chromitites in a Archean-Paleoproterozoic greenstone belt in
Brazil. Raman analyses showed that methane in chromitites is adsorbed
along microfractures in samples enriched with chromium and ruthe­
nium, two powerful catalysts of Sabatier reaction. Hence, the hypothesis
that chromitites may act as a gas source rock (Etiope et al., 2018; Etiope
and Whiticar, 2019).
Concerning the Samail ophiolite, Nothaft et al. (2021) claimed that
chromitites cannot be the source of the methane observed in the wells,
because massive chromitites do not exist in the region as they are not
reported in the cores or drill cuttings from any well in the area, or in
outcrops in the surrounding area. This is not correct. The authors missed
the studies with chromitite mapping and PGE (Platinum Group Ele­
ments) analyses of Page et al. (1982) and Rollinson and Adetunji (2015).
Page et al. (1982), in particular, report at least eight chromitite outcrops
(where PGEs were observed) in the area investigated by Nothaft et al.
(2021); one outcrop is 5 km far from the NSHQ14 well (Fig. S2).
Chromitites, containing PGE, in the Ibra area correspond to the basal
ultramafic member (predominantly harzburgite and dunite tectonite),
which develops underground in other portions of the Wadi Tayin Massif
(Page et al., 1982; Rollinson and Adetunji, 2015).
Moreoever, Nothaft et al. (2021) assumed that there is no free H2
available for CO2 hydrogenation, as the subsurface is water-saturated.
However, underground rocks are not always water-saturated. Any
aquifer does have a bottom level. Examples of water-free rock systems
include gas-filled fractures and pores observed: a) in petroleum fields, b)
in 3D microscopy of immiscible displacement in rocks (Porter et al.,
2015) and c) in gas migration studies of gas columns, slugs and bubbles
along fracture networks (e.g., Malmqvist and Kristiansson, 1984). In
stagnant, serpentinized systems, Sleep et al. (2004) postulated that
under certain conditions H2 fugacities can exceed saturation. H2(gas)
bubbles and gas-filled fractures could exist under such conditions.
Nothaft et al. (2021) do not mention that ~5 km from the well CM2A,
there is a H2-rich bubbling spring, named Lauriers Roses, or Nasif
(Fig. S2). This site releases free gas with 61% of H2, 23% of N2 and 15%
of CH4 with δ13C–CH4: +7.9‰ (Vacquand et al., 2018). Not considering
this site, as well as the numerous springs and bubbling seeps that are
widespread in the Samail ophiolite, and which have variable N2–H2–CH4
compositions, makes any interpretation uncontextualized.
The availability of CO2 should also be examined in the regional
context. While CO2 does not (obviously) exist in the hyperalkaline fluids
intercepted by the wells, it can be pervasive in the metasedimentary
rocks below the ophiolitic nappe, and migrate upwards through the
numerous fault and fracture systems of the region (Vacquand et al.,
2018). These fracture systems can be the loci of H2 and CO2 mixing. It is
worth noting that, as mentioned in Section 5.2.3, 13C-enriched CO2 may
be derived by thermal decarbonation from the 13C-enriched Permian
carbonates, such as the Hawasina, Sumeini, or Saih Hatat formations
occurring below and in lateral tectonic contact with the Samail ophiolite
nappe. Hydrogenation of 13C-enriched CO2 may then lead to
13
C-enriched methane.
5.4. Gas outside the observed aquifer
The assumption behind the hypothesis of a microbial origin is that
CH4 is mostly produced within the peridotite aquifer. A wider look at the
regional context in Oman reveals that the gas seepage system extends
beyond the local peridotite aquifer of the Wadi Tayin Massif (Fig. S3).
For example, N2 is another missing item in the analyses of the Samail
ophiolite wells in Nothaft et al. (2021). N2-rich springs are abundant in
the region (e.g., Boulart et al., 2013; Vacquand et al., 2018); in these
springs N2 is mostly of crustal, sedimentary origin, with a minor atmo­
spheric component, as evidenced by δ15N isotopes (Vacquand et al.,
G. Etiope and C. Oze Applied Geochemistry 143 (2022) 105373

Fig. 5. Flow chart showing how the several techniques and analyses may guide the unravelling of methane origin.

2018). Basically, N2 comes from sediments below the ophiolitic units, reservoir model would be compatible, and a microbial origin could be
from clay dewatering or destabilization of ammonium bearing clays. The considered only if methanogens use a 14C-free substrate. On continents,
existence of crustal degassing is then suggested by relatively high con­ potential C feedstock for methanogens are carbonate veins, typically
tents of 4He (Vacquand et al., 2018). This testifies that the gas seepage pervading the peridotites (Suzuki et al., 2014), atmospheric CO2 and the
system crosses the whole Samail ophiolitic nappe and includes deep products of present-day serpentinization, formate (HCOO− ), acetate
sedimentary inputs. With respect to the CH4-rich serpentinized ultra­ (CH3COO− ) and CO (Miller et al., 2018; Nothaft et al., 2021). In the
mafic complex of Hakuba-Happo in Japan, a similar approach Samail ophiolite, both carbonate dissolved in groundwater and car­
combining noble gases and carbon isotopes was applied to identify bonate veins have measurable 14C content (Abdalla et al., 2018; Nothaft
subsurface fluid components and ultimately identify crustal degassing as et al., 2021 and references therein). If methane is 14C-free, carbon in
the dominant carbon source (Suda et al., 2022). Can we exclude that the water and carbonate veins cannot represent a substrate for metha­
sedimentary input below the Samail ophiolite, in addition to N2, may nogens; then formate, acetate and CO feeding the microbes should be
also bear CO2 and hydrocarbons into the overlying ultramafic rocks? “ancient” and 14C-free, thereby, contributing to the dissolved carbon in
14
C-free aquifers, which have not been identified so far (Abdalla et al.,
14 2018). Analysis of 14C–CH4 is a high priority target as it may help clarify
5.5. Is Samail ophiolite methane C-free?
the origin of methane in many cases.
A key and missing parameter, not investigated so far in the Samail
6. Concluding remarks
gas, is the amount of radiocarbon (14C) in CH4. Measurable amounts of
14
C indicate that methane is formed from a “modern” C precursor, while
This review is meant to stress the importance of examining gas in
absence of 14C (expressed as 0 percent of modern carbon) means that the
ultramafic rock systems with a holistic approach, considering a wider
CH4 carbon is older than ~50,000 years (fossil C). 14C–CH4 analyses in
geological-geochemical (regional to global) context, with a vision that
several CH4-discharge serpentinization sites, Zambales (Philippines),
goes beyond the local observation. Specifically, the attribution of an
Ronda (Spain), Chimaera (Turkey), Acquasanta (Voltri, Italy), Happo
origin to a certain gas observed in a given system cannot be solely based
(Japan) and in the submarine Lost City (Abrajano et al., 1990; Pros­
on the data observed in that system, but shall consider what is observed
kurowski et al., 2008; Etiope and Schoell, 2014; Etiope et al., 2016;
in other geologically and geochemically similar places and, in particular,
Etiope and Whiticar, 2019; Suda et al., 2022), always revealed that
the elevated migration potential of gas, because the genetic history of a
methane is 14C-free. In contrast, the carbon precipitated or dissolved in
gas is often untied from the environment where it is sampled. The three
the related hyperalkaline springs contains substantial 14C, dating the
main theories of methane’s origin in continental ultramafic rock systems
water circulation of several hundred to a few thousand years (Cipolli
(i.e., abiotic CO2-hydration-reservoir model, fluid inclusion model and
et al., 2004; Marques et al., 2018; Etiope et al., 2016; Suda et al., 2022).
microbial-aquifer model) have their pros and cons. Some geochemical
Therefore, methane cannot be sourced from any of the 14C-bearing
data, taken individually, can be compatible with all three models, but
carbon dissolved in hyperalkaline waters.
some models are clearly incompatible with other data (Fig. 4). In
In the Samail ophiolite, 14C–CH4 has yet to be measured. If methane
particular:
has modern C, with measurable 14C, the deep origin models (i.e., fluid
inclusion and CO2 hydrogenation-reservoir models) would not be suit­
- methane clumped-isotopes are ambiguous genetic proxies for abiotic
able, as they consider a geological C precursor. The microbial origin
gas produced at moderate temperature: this gas is typically in iso­
hypothesis, with methanogens using modern C substrates, would be
topologue disequilibrium, and there is strong possibility that FTT
compatible. If methane is 14C-free, like in other continental serpentini­
reactions in natural systems give low/negative values of Δ13CH3D vs
zation sites, then both the fluid inclusion and CO2 hydrogenation-

10
G. Etiope and C. Oze
Δ12CH2D2 (as observed in laboratory) undifferentiated from
microbialgenesis.
- Radiocarbon analysis of methane is a fundamental step in the
interpretation of gas origin, as it precludes microbial origin from
groundwater that contain 14C. Radiocarbon-CH4 analyses are
required in more sites, and models and analyses that may verify
whether methanogenes may use a14C-free substratum need to be
developed.
- The existence of gaseous alkanes heavier than methane (i.e., ethane,
propane and butane) must be interpreted considering that these
gases can be affected by molecular fractionation processes during
migration, including differential solubility. Additionally, a wide
range of alkane composition can be produced by FTT processes,
depending on temperatures, catalysts and time evolution (e.g., by
polymerization); therefore, their relative composition (specifically
the C1/(C2+C3) ratio) observed in shallow aquifers or far from their
putative source rocks, cannot be used as a genetic index.
Fig. 5 is a flow chart of holistic interpretation of data, suggesting how
the several techniques and analyses may guide the unravelling of
methane origin. The major constraint in any interpretation of methane
origin is the capability to sustain a continuous gas flow system, in terms
of gas emission intensity, longevity and spatial extension. In this respect,
the CO2 hydrogenation - reservoir model, with the recognition of source
rocks and reservoir rocks, similar to sedimentary natural gas systems
and related seeps, can explain this aspect. The fluid inclusion and aquifer
microbial models presently fail to explain how gas pressure, necessary to
explain the surface emissions, can develop. Echoing Vacquand et al.
(2018), Etiope and Whiticar (2019), and Monnin et al. (2021), metha­
nogens collected in the wells are not necessary representative of the
conditions of methane generation at depth. Microbialgenesis might be a
surficial process, while methane may mostly originate outside the
investigated aquifer. While CH4-bearing fluid inclusions may occur in
continental peridotites, the existence, everywhere, of large volumes of
inclusions necessary to continuously sustain the various gas discharges
(bubbling seeps and high amounts of dissolved CH4 transported by
springs) needs to be verified. For this model, there needs to be a justi­
fication and evidence for explaining how inclusions should be spatially
distributed and liberated in a way to produce, for long times, the volu­
minous surface output of the gases observed. Additional complexity and
interpretative challenges arise when minor secondary gas sources are
involved and fluids mix during their migration to the surface. However,
in several study areas beyond Oman (Table S1), the origin of methane
can be clarified or confirmed by adding some missing analyses following
the approach proposed in Fig. 5 (e.g., radiocarbon in the springs in
California and Canada; fluid inclusions in the ophiolitic rocks at the
seeps in Turkey and Japan; microbial analysis in the Ronda ophiolite
springs in Spain).
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
This work has benefitted from discussions with Edward Young,
Naizhong Zhang and Stefano Salvi.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.apgeochem.2022.105373.
11
Applied Geochemistry 143 (2022) 105373
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