The deep, hot biosphere: Twenty-five years 
of retrospection
Authors: Daniel R. Colman, Saroj Poudel, Blake W. 
Stamps, Eric S. Boyda, and John R. Spear
This is a postprint of an article that originally appeared in Proceedings of the National Academy 
of Sciences on July 3, 2017.
Colman, Daniel R. , Saroj Poudel, Blake W. Stamps, Eric S. Boyd, and John R. Spear. "The 
deep, hot biosphere: Twenty-five years of retrospection." Proceedings of the National Academy 
of Sciences 114, no. 27 (July 2017): 6895-6903. 
DOI: 10.1073/pnas.1701266114.
Made available through Montana State University’s ScholarWorks 
scholarworks.montana.edu 
The deep, hot biosphere: Twenty-five years
of retrospection
Daniel R. Colmana, Saroj Poudela, Blake W. Stampsb, Eric S. Boyda,c,1, and John R. Spearb,c,1
Edited by David M. Karl, University of Hawaii, Honolulu, HI, and approved May 16, 2017 (received for review January 25, 2017)
Twenty-five years ago this month, Thomas Gold published a seminal manuscript suggesting the presence of a
“deep, hot biosphere” in the Earth’s crust. Since this publication, a considerable amount of attention has
been given to the study of deep biospheres, their role in geochemical cycles, and their potential to inform on
the origin of life and its potential outside of Earth. Overwhelming evidence now supports the presence of a
deep biosphere ubiquitously distributed on Earth in both terrestrial and marine settings. Furthermore, it has
become apparent that much of this life is dependent on lithogenically sourced high-energy compounds to
sustain productivity. A vast diversity of uncultivated microorganisms has been detected in subsurface envi-
ronments, and we show that H2, CH4, and CO feature prominently in many of their predicted metabolisms.
Despite 25 years of intense study, key questions remain on life in the deep subsurface, including whether it is
endemic and the extent of its involvement in the anaerobic formation and degradation of hydrocarbons.
Emergent data from cultivation and next-generation sequencing approaches continue to provide promising
new hints to answer these questions. As Gold suggested, and as has become increasingly evident, to better
understand the subsurface is critical to further understanding the Earth, life, the evolution of life, and the
potential for life elsewhere. To this end, we suggest the need to develop a robust network of interdisciplin-
ary scientists and accessible field sites for long-term monitoring of the Earth’s subsurface in the form of a
deep subsurface microbiome initiative.
geobiology | biogeochemistry | hydrogen | subsurface | thermophiles
A quarter-century ago this month, Thomas Gold, an
Austrian-born astrophysicist from Cornell University, pub-
lished a paper in these pages entitled simply, “The deep,
hot biosphere” (1). In this paper, followed by a book of
the same title (2), Gold suggested that microbial life is
likely widespread throughout Earth’s subsurface, residing
in the pore spaces between grains in rocks. Furthermore,
he speculated that this life likely exists to a depth
of multiple kilometers, until elevated temperature
becomes the constraining factor. Gold hypothesized
that life in subsurface locales is supported by chemical
sources of energy, rather than photosynthetic sources,
upon which surface life ultimately depends (1). The nu-
trients that support this subsurface life are provided by
both the migration of fluids from the depths of the
Earth’s crust and the host rock itself, which contains
both oxidized and reducedminerals. Although it is likely
to be all microbial, Gold posited that the mass of sub-
surface life in this little-known biosphere was compara-
ble to that present in surface environments. Gold
thought that if there is life at depth, the rocks that have
(or could produce) “hydrogen (H2), methane (CH4), and
other fluids. . . would seem to be the most favorable
locations for the first generation of self-replicating
systems,” keenly aware that “such life may be widely
disseminated in the universe” (1). Moreover, Gold
hypothesized that hydrocarbons and their derived
products fuel chemosynthetic subsurface life and that
these hydrocarbons are not biology reworked by ge-
ology, but, rather, geology reworked by biology (2).
Although he did not have a doctorate, Gold (1920–
2004) was highly recognized as a scientist, as evidenced
by receiving a Gold Medal of the Royal Astronomical
Society (1985) and a Humboldt Prize (1979); member-
ship in the National Academy of Sciences (1974); and
induction into the American Academy of Arts and Sci-
ences (1974), the Royal Society (1964), the American
Geophysical Union (1962), and the Royal Astronomical
Society (1948), among others. As an author of ∼300
scholarly papers, Gold had a penchant for contributing
his thoughts to fields well beyond his own of astro-
physics. In the foreword to Gold’s book The Deep Hot
aDepartment of Microbiology and Immunology, Montana State University, Bozeman, MT 59717; bDepartment of Civil and Environmental Engineering,
Colorado School of Mines, Golden, CO 80401; and cThe NASA Astrobiology Institute, Mountain View, CA 94035
Author contributions: D.R.C., E.S.B., and J.R.S. designed research; D.R.C., E.S.B., and J.R.S. performed research; D.R.C., S.P., B.W.S., E.S.B., and
J.R.S. analyzed data; and D.R.C., B.W.S., E.S.B., and J.R.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence may be addressed. Email: jspear@mines.edu or eboyd@montana.edu.
Biosphere, the theoretical physicist Freeman Dyson wrote,
“Gold’s theories are always original, always important, usually
controversial—and usually right” (2). Stephen Jay Gould consid-
ered Gold as “one of America’s most iconoclastic scientists” (3).
Gold’s colleague and frequent coauthor Hermann Bondi, whom
Gold met as a fellow “enemy alien” in an internment camp at the
start of the Second World War, penned that, “Thomas Gold was a
most unusual scientist. Brilliant in his ideas, unconstrained in the
range of his interests, rock solid in his use of the fundamental laws of
physics to make the most surprising deductions from them, making
himself at home in fields of science he had no connections with
before. . .” (4). Building on this sentiment, astronomer Steve Maran
commented, “Unlike most scientists who are content to pursue the
advancement of knowledge in small, incremental steps, Gold came
up with new ideas by starting from the original principles” (3). Gold
himself said, “In choosing a hypothesis there is no virtue in being
timid. . .(but) I clearly would have been burned at the stake in an-
other age” (3). Gold also took issue with the limitations of peer
review, where established scholars pass judgment on new ideas
and new papers before publication, which in turn rewards incre-
mental steps, but few bold ideas that provide breakthroughs. Evi-
dence of the effects of peer review, in Gold’s view, comes from the
sheer volume of great ideas that came out of the first half of the
20th century relative to the latter half (3).
Gold believed that biology is just a branch of thermodynamics,
being supported by energy in disequilibria in chemical reactions
that would otherwise equilibrate if they were not held up by
kinetic or mechanistic traps (5). To this end, the chemical energy
available inside a planetary body would have been the first energy
source, and solar energy, carbon fixed through photosynthesis,
was a later adaptation of life (3). Although revolutionary in the
infancy of the proposal, and not entirely unfounded in the scien-
tific literature (6), informatics, geochemical, and isotopic evidence
now provide evidence to support this view (7). Controversially,
Gold also believed that oil and gas, which have fueled modern
industrial nations for the past century, were born out of the Big
Bang and were incorporated into the Earth’s crust at the time of
the Earth’s coalescence 4.5 billion years ago. Gold arrived at this
conclusion from the humble observation that meteorites and
other planetary bodies contain abundant and compositionally di-
verse hydrocarbons (e.g., the methane lakes of the Jovian moon
Titan), begging the question as to why hydrocarbons in the deep
subsurface of Earth need to have a different origin (2). Bondi
thought that this proposal in geology was Gold’s most important
intervention to a field outside his own (4). Although speculation
surrounding the formation of oil and gas has resulted in significant
debate within the geosciences, the idea of abundant abiogenic
hydrocarbons percolating upward had perhaps an even more pro-
found influence on the biosciences by providing impetus to un-
derstand how such compounds could fuel a deep, hot biosphere
since early in Earth history. Much of the debate concerning the
presence of mantle-sourced hydrocarbons has been settled since
Gold’s initial proposals, including evidence that mantle-sourced
hydrocarbons in crustal environments are likely to be far less abun-
dant than originally proposed (8, 9).
Regardless of present-day evidence indicating that light hydro-
carbons in the crust are unlikely to have originated in the mantle,
Gold pioneered the notion that such hydrocarbons (regardless of
source) could sustain life in the subsurface to known depths of
10 km and possibly down to 300 km, if the temperature was below a
hypothetical life-maximum of 150 °C (10). At higher temperatures,
deeper within the crust/mantle, “cracking” of hydrocarbons could
release volatiles such as hydrogen (H2) and methane (CH4), which
could percolate upward to thermal regimes that allow for life,
thereby providing fuel. If true, H2 or CH4 sourced from abiogenic
hydrocarbons, and the life that is dependent on these compounds,
could be widespread in the subsurface of not just Earth, but also
other planetary bodies (11–13). Indeed, H2 has become a major
focal point for microbiologists studying the deep subsurface (14,
15) and has been suggested to be the fuel that supported the
earliest forms of life on Earth (16–18).
Gold’s deep, hot biosphere contribution challenged para-
digms in subsurface science, petroleum research, the origin and
evolution of life, and the search for life on other planets. Some
argue that aspects of his ideas are highly flawed (e.g., the source
of hydrocarbons in subsurface environments), whereas others ar-
gue that his ideas and the concept of a deep, hot biosphere make
perfect sense. Regardless of one’s personal opinion, Gold’s ideas
on the deep, hot biosphere have had a tremendous impact on
scientific discourse, with the article being cited >325 times (Web
of Science), and the book remains a top seller on Amazon.com.
Fig. 1 shows how the work continues to be highly cited by numer-
ous fields outside of his field of astrophysics, indicative of its broad
reach. Here, we provide an overview of new insights into the
deep, hot biosphere developed over the past 25 years. These
insights were made possible by the development of new tools
and technologies and increased and better access, as well as en-
larged interest—all of which can be traced (at least in part) to Gold’s
pioneering paper. Particular emphasis is placed on the nature of
microbial communities in subsurface water–rock-hosted ecosys-
tems and their extent and diversity, and new insights into processes
that sustain this life.
Fig. 1. Citation network of Gold’s 1992 “The deep, hot biosphere”
publication (1). The citation index for Gold (1992) was downloaded
from the ISI Web of Knowledge database (apps.webofknowledge.
com/) and consists of 327 citations as of November 15, 2016.
Citations were grouped by subject fields, or Web of Science
Categories (WC). If multiple fields were given for a citation, all were
considered in the network. Publications are shown as colored nodes,
where Gold’s publication is the large center-most black node, and the
others are colored according to year of publication (scale given on
right) and sized according to the number of citations each has
received (log scale given on right). Publications are grouped by
subject field (black nodes), and those with more than five publications
are labeled. A force-directed layout in Cytoscape (Version 3.2.0) was
used to generate and view the citation network.
Energy at the Interface Between the Biosphere and the
Geosphere
Gold controversially suggested that hydrocarbons originating in
the subsurface (particularly from the mantle) or substrates derived
from these hydrocarbons provide a source of energy capable of
supporting a deep, hot biosphere. Although the merits of this
hypothesis have been heavily scrutinized and largely proven false
(discussed below), it is now abundantly clear that microbial life
inhabits the subsurface and appears to be generally dependent
on lithogenic substrates produced from water–rock interactions.
H2 and CH4 derived from mantle sources are now known to con-
tribute minimally to their overall abundance in deep terrestrial
subsurface settings (8, 9), but, importantly, Gold’s ambitious
treatise set forth a generation of researchers that have now
documented the nature of subsurface biospheres and the geo-
logically sourced energy substrates that sustain them. Gold rightly
drew comparisons from then-recent observations at hydrothermal
vents that chemical disequilibria involving H2 and CH4 supported
a diversity of chemosynthetic microorganisms that may also exist
at depth in fracture zones. H2, in particular, has drawn consider-
able attention in the following 25 years as a major reductant for
chemosynthetic life and is now known to be predominantly
abiologically sourced via (i) radiolytic splitting of aqueous frac-
ture fluids in granitic rocks (19–23); (ii ) reduction of water by
iron-bearing minerals in basalts and peridotites (14, 15); and
(iii) radical-based splitting of water driven by the physical shearing
of silicate minerals through a “mechano-radical” mechanism (24,
25) (Fig. 2). It is also possible that some H2 supporting subsurface
lithoautotrophic primary production derives from H2 produced at
depth by processes akin to those suggested by Gold 25 years
ago. Regardless of the mechanism of formation, this lithogenic H2
is abundant in Precambrian terrestrial formations (23, 26) and fuels
ecosystem level primary productivity both in subsurface and sur-
face environments impacted by subsurface processes such as
geothermal and hydrothermal systems (27–30).
The ability to metabolize H2 is pervasive in the microbial world,
with ∼30% of taxa with available genomes encoding for [FeFe]-,
[NiFe]-hydrogenase, or [Fe]-hydrogenases (31), the principal en-
zymes involved in H2 metabolism (31). An analysis of [FeFe]- and
[NiFe]-hydrogenase homolog abundances in community ge-
nomes from subsurface environments reveals nearly an order of
magnitude more copies per mega-base pair of sequence relative
to those from surface environments (Fig. 3), consistent with the
central role of H2 in the energy metabolism of subsurface life.
Importantly, high concentrations of H2 can also drive the abiotic
hydrogenation of CO2 through a process sometimes referred to as
Fischer Tropsch synthesis (32). Indeed, at high temperatures, hy-
dration of olivine and the H2 produced has been shown to cata-
lyze the abiotic reduction of aqueous CO2 to formate (HCOO
−),
which can then equilibrate to carbon monoxide (CO) (33). In-
triguingly, genomes of subsurface microbial communities are also
enriched in genes allowing for the use of HCOO− and CO under
anoxic conditions, formate dehydrogenase and nickel-dependent
CO dehydrogenase, respectively, compared with genomes
from surface communities (Fig. 3). In contrast, molybdenum-
dependent CO dehydrogenase was not found to be enriched in
subsurface communities relative to surface communities, which
may be due to limited Mo availability in anoxic and possibly sulfidic
conditions present in many subsurface locales (34).
Methanogens, organisms with metabolisms suggested to be
the most “primitive” among those available in extant life (18, 35),
combine H2 with CO2 to drive energy metabolism and produce
CH4 as a metabolite. Methanogens use an ancient pathway of
CO2 fixation termed the reductive acetyl CoA pathway (35), which
involves a number of Ni- and Fe-dependent enzymes. Chief
among these is the acetyl CoA-synthase (ACS), which is also
enriched in subsurface microbial communities relative to those
found in surface environments (Fig. 3). The acetyl CoA pathway
proceeds along a reaction pathway that involves a number of in-
termediates, namely, CO and HCOO−, that also can be produced
during the abiotic hydrogenation of CO2 in the production of CH4
and other hydrocarbons (33). Minerals that catalyze the abiotic
reduction of CO2 often contain copious amounts of Fe and Ni,
similarities that have led to the hypothesis that the emergence of
biological catalysts (e.g., CO-dehydrogenase, ACS, and [NiFe]-
and [FeFe]-hydrogenase) involved in CO2 reduction via H2 took
place in a subsurface environment where these minerals were
abundantly available (35, 36). The most primitive methanogen by
phylogenetic reconstruction is Methanopyrus kandlerii (37), a
hyperthermophilic organism isolated from a deep sea hydro-
thermal vent that grows at temperatures up to 122 °C (38). The
physiological characteristics of this organism, its ecology, and
its primitive nature are consistent with the notion of a deep,
hot biosphere.
The Diversity of Life in the Deep Subsurface
Initially, we learned about the deep, hot biosphere from cultivation
of taxa in fluids sampled from oil wells, mines, caves, and geo-
thermal and hydrothermal environments, among others (39–42).
Since Gold’s call to probe the deep biosphere, increased access
and sampling of subsurface environments in terrestrial and marine
environments through drilling projects and increased collaboration
with industrial partners have resulted in substantial expansion of the
known microbial taxa in these environments. Furthermore, appli-
cation of molecular approaches has resulted in the discovery of a
plethora of previously unknown microbial lineages, particularly
within the archaeal domain (43–49). Although a precise definition of
what depth below the surface is considered “deep” is system-
dependent, constraining the limits of the deep, hot biosphere
to those environments that exhibit temperatures higher than
∼50 °C and are >1 m below the surface [as is used elsewhere to
operationally define the “deep subsurface” (50)] provide a
metric from which to compare like environments.
Although there are a number of similarities between marine
and terrestrial subsurface environments, important differences
Fig. 2. Generalized schematic of the subsurface crustal environment
and interacting forces that produce habitable conditions in the
subsurface. Inset shows a magnified view of the interactions
occurring at the surface–subsurface interface, and mechanisms of H2
production in the subsurface that are discussed in the text are
highlighted.
also exist. Most conspicuously, the abundance of dissolved or-
ganic carbon (DOC) that is available to support heterotrophic
populations differs between the two. The burial of photosyn-
thetically derived organic carbon in marine subsurface environ-
ments supports heterotrophic populations at significant depths
(51, 52), whereas studies of the terrestrial subsurface have largely
focused on systems that are generally removed from surface
influences. Although much of the deep marine sediments re-
covered from drilling programs are below the thermal temper-
atures that we define as hot (50 °C), recent estimates have
suggested that ∼34% of total ocean sediment volume exists in the
temperature range occupied by thermophilic microorganisms
(40–100 °C), with an additional 5% at temperatures theoretically
capable of supporting life [100–120 °C (53)]. Clearly, the definition
of deep is contextually dependent and has consequences for the
physicochemical characteristics of such systems. Regardless, the
defining features of environments hosting a deep, hot biosphere
are that they are removed from surface photosynthetic processes
by at least one or more steps (following ref. 50), have elevated
temperature, and are generally anoxic. Although a number of
other definitions can be used, this framework is used to guide
the following discussion of the deep, hot biosphere.
In terrestrial settings, studies within mines (43, 45, 54, 55) at
depths of between 102 and 103 m below the surface and borehole
drilling into hydrothermal environments (15) have provided critical
information on the nature of the deep, hot biosphere. Likewise,
ocean-drilling programs in areas associated with hydrothermal
activity have provided equally important insight into the nature of
life in deep, hot marine environments (44, 48, 56–59). These
studies reveal a unique and considerable diversity of both culti-
vated and uncultivated lineages of Bacteria and Archaea that
predominate deep thermal subsurface environments. Archaea
that were detected in subsurface mine samples in South Africa
and Japan include representatives from candidate divisions
that have now been given the epithets of “Bathyarchaeota,”
“Hadesarchaea,” and “Aigarchaeota” (59–61). Deep borehole
fluids from Idaho and South Africa contained significant pop-
ulations of thermophilic, methanogenic Archaea (15, 62). Con-
siderable bacterial diversity has also been detected in thermal
subsurface environments and includes members of the Aquificae,
Firmicutes, δ-Proteobacteria, and Nitrospirae, among other pro-
teobacterial groups and numerous uncultivated candidate divi-
sions (46, 49, 62–66). Similar microbial taxa have been observed in
deep, hot marine subsurface systems in addition to Archaea from
the Archaeoglobales, Thermococcales, and anaerobic methano-
trophic archaea (ANME) lineages and bacteria related to Thermo-
togae, Chloroflexi and the “Aminecenantes” (formerly OP8),
among others (44, 48, 56–58). Although the previous 25 years of
work have revealed abundant, novel taxonomic diversity in deep,
hot subsurface environments, accompanying physiological
diversity was less forthcoming until recent developments in high-
throughput, -omics–based approaches.
Omic Insights into the Physiological Capacity of the Deep
Subsurface Microbial Biosphere
The oligotrophic nature of subsurface environments selects for
microorganisms that thrive on minimal energy gradients pro-
vided by available redox couples in highly reducing, low-oxidant
environments. A subset of these environments that are removed
from surface carbon input have been collectively termed sub-
surface lithoautotrophic microbial ecosystems (14). A lack of
available surface-derived DOC likely necessitates a general ca-
pacity for autotrophy that is fueled by lithogenic energy sources,
such as abiogenic H2. The prevalence of methanogenic Archaea
in many of these systems is not surprising, because the H2/CO2
redox couple for biomass production typically dominates envi-
ronments with minimal energetic gradients (67).
The prevalence of autotrophic sulfate-reducing archaea and
bacteria (SRA/B) is consistent with the thermodynamic favorability
of the H2/SO4
2− redox couple in the deep subsurface and the
availability of these compounds for use. Key differences in the
origin of SO4
2− in marine and terrestrial subsurface settings exist,
but its prevalence nevertheless leads to commonalities in the
physiologies that support microorganisms in these environments.
For instance, in the terrestrial subsurface, SO4
2− is likely produced
via oxidation of pyrite by radicals produced during radiolytic
splitting of water—a process that was recently shown to play a key
role in supporting sulfur-based microbiomes in the South African
subsurface (68). In contrast, marine sediments and the underlying
bedrock feature an abundance of sea-water–derived SO4
2−
(originally from weathering of continental sulfides). Indeed, SO4
2−
is a predominant electron acceptor in anoxic marine subsurface
sediments (52). Like methanogens, autotrophic SRA/Bs fix CO2
using the acetyl CoA pathway and also tend to use formate and
CO as carbon and/or electron donors (69). The prevalence of key
proteins involved in H2, CO, formate and the acetyl CoA pathway
of CO2 fixation encoded in the genomes of subsurface commu-
nities (Fig. 3) relative to surface communities is consistent with
these observations.
Gold suggested that if the “archaebacteria” (now Archaea)
were indeed the most primitive of organisms and were largely
0
2
4
6
8
[FeFe]-Hydrogenases
0
10
20
[NiFe]-Hydrogenases
0
20
40
Mo-CO Dehydrogenases
0
2
4
6
8
Ni-CO Dehydrogenases
0
2
4
6
8
Acetyl-CoA Synthases
0
10
20
Formate Dehydrogenases
M
ea
n 
H
om
ol
og
ue
 C
ou
nt
 / 
M
bp
 (x
 10
-
4 )
Su
rfa
ce
Su
bu
rfa
ce
Su
rfa
ce
Su
bu
rfa
ce
Fig. 3. Average abundances of key metabolic proteins in surface and
subsurface metagenomes. Average abundances are given as the
number of positive BLASTp searches found for each environmental
metagenome in NCBI’s metagenome ftp server (ftp://ftp.ncbi.nlm.
nih.gov/genomes/genbank/metagenomes/). Search bait sequences
for each functional gene were used after empirically validating the
appropriate E-value cutoff that would only include homologous
protein sequences in search results. Average abundances were
normalized by the total sequence length (in base pairs) of each
metagenome. Metagenomes without any hits to the six functional
genes were not included in the results. Companion metadata were
used to group metagenomes into surface and subsurface categories.
Only environmental (e.g., nonengineered and non-host-associated)
metagenomes were used in the analyses. Metagenomes were
classified as surface or subsurface based on available metadata in
which subsurface environments were defined as groundwater, oil
reservoir, rock porewater, or marine sediments > 1 m below sea
floor, and surface environments included all others.
thermophilic, that they may have “evolved at some depth in the
rocks . . . spread laterally at depth, and they may have evolved and
progressed upwards to survive at lower temperatures nearer the
surface” (1). It is intriguing to note then that the current revolution
in archaeal genomics is, in part, driven by the discovery of nu-
merous archaeal “subsurface” lineages, many of which provide
key insight into the evolution of all Archaea, if not all life. Meta-
bolic reconstructions from genomic representatives of several
Archaea, initially detected in subsurface gold mines, have pro-
vided support for the importance of autotrophy and H2/C1 (one-
carbon compounds) metabolism. Candidatus (Ca.) Caldiarchaeum
subterraneum of the proposed Aigarchaeota division (formerly
the hot water crenarchaeotic group I) was proposed to be capable
of autotrophy in conjunction with H2 and CO as electron sources
and NO3
− and/or O2 as electron acceptors (60). Additional
genomic comparisons incorporating representatives of the
Aigarchaeota division from alkaline hot springs provide further
evidence for the capacity of autotrophic metabolism (70), al-
though some members may also be heterotrophic (70, 71), and it
is unclear whether their [NiFe]-hydrogenases are responsible for
H2 oxidation or maintenance of cell redox state (71). Likewise,
genomic analysis of members of the Hadesarchaea (formerly the
South African gold mine euryarchaeotic group) suggests that they
are also reliant on H2 and/or CO as an energy source linked to
NO2
− reduction and capable of autotrophy via the acetyl CoA
pathway (61). Lastly, the Bathyarchaeota (formerly the mis-
cellaneous crenarchaeotic group) have received considerable
attention because of mesophilic representatives distributed
ubiquitously in ocean and freshwater sediments (72, 73).
The presence of protein complements necessary for meth-
anogenesis or methane oxidation (including the methane-
activating McrA protein) in the first nearly complete genomes of
the Bathyarchaeota hinted at the potential for CH4-based me-
tabolism (74). Analyses of other genomes from marine sediments,
however, have suggested a role for acetogenesis in supporting
these organisms (75). Additionally, a recent analysis of group 1
ANME (ANME-1) suggests that their McrA proteins are involved in
the oxidation of short-chain alkanes such as butane (76). The McrA
protein sequences of the Bathyarchaeota (and Hadesarchaeota)
are more closely related to the McrA of ANME-1, rather than
methanogens, suggesting that these organisms may actually be
involved in short-chain alkane degradation, rather than methano-
genesis. Although the exact role of the Bathyarchaeota in sub-
surface carbon cycling remains unclear, the capacity for autotrophy
and a reliance on H2 is consistent with other subsurface Archaea.
Clearly, further cultivation efforts and physiological-based studies
are needed to assess the role of these lineages in light hydro-
carbon and H2 cycling in subsurface environments. Neverthe-
less, the putative capacity for autotrophy and the use of H2 and/
or CO appear to be common among the Archaea present within
high-temperature subsurface environments and are consistent
with Gold’s suggestions 25 years ago.
In addition to the aforementioned Archaea, studies over the
previous 25 years have documented the prevalence of thermo-
philic sulfate-reducing bacteria (SRB) as key members of deep, hot
terrestrial and marine biospheres (46, 62, 77–79). The discovery
and genomic characterization of an autotrophic sulfate-reducing
Firmicutes bacterium, Ca. Desulforudis audaxviator, has pro-
vided valuable insight into life in the deep, hot subsurface. Ca.
D. audaxviator dominates microbial communities at depths up to
2.8 km in the South African Witwatersrand Basin (46, 77). Genomic
reconstruction suggests that oxidation of H2 and formate coupled
with reduction of SO4
2− is likely to drive inorganic carbon and
nitrogen fixation in microbiomes dominated by Ca.D. audaxviator
(46). The lack of evidence for significant auxotrophy suggests that
Ca. D. audaxviator contains the genomic machinery necessary for
maintaining a self-sustainable deep subsurface ecosystem (46).
Although it was originally proposed that Ca. D. audaxviator
comprises a single-species, self-sufficient microbial community,
both earlier 16S rRNA gene surveys (77) andmore recent genomic
analyses have indicated that additional bacterial taxa are present
in the fracture waters and that it is an evolutionarily and ecologi-
cally dynamic system with considerable horizontal gene transfer
(HGT) and viral infection (66). The genome of Ca. D. audaxviator
encodes for six and three phylogenetically distinct [FeFe]- and
[NiFe]-hydrogenases (31), respectively, further underscoring the
importance of H2 in its metabolism and suggesting an important
role for HGT in its evolution. Analyses of high-temperature crustal
fluids of the subsurface basement rock in the flanks of the Juan De
Fuca Ridge spreading center have also indicated a prevalence of
SRA/Bs (78–81), where organisms related to Ca. D. audaxviator
and other SRA/Bs are dominant. Recent metagenomic analyses of
these samples have provided a glimpse into the considerable
phylogenetic and metabolic diversity of the deep, hot marine
subsurface via reconstructed genomes from metagenomes that
have expanded on previous molecular surveys (81).
Although much insight has been gained into the nature of life
in the deep, hot biosphere over the previous 25 years since
Thomas Gold’s seminal work, there are a number of questions that
remain unanswered. For instance, what is the role of syntrophy
in sustaining and enabling subsurface microbial ecosystems? Re-
cent analyses of 1.34-km-deep warm fracture fluids from the
Witwatersrand Basin suggested a putative role of syntrophic in-
teractions in sustaining a diverse community that is fueled by CH4
(68). Community-wide trophic interactions are likely integral to
the functioning of these isolated systems and warrant further
functional-based studies. Another paramount question is whether
phylotypes that belong to clades that are distributed in both
subsurface and surface environments contain unique physiologi-
cal adaptations that could provide insight into the selective
pressures imposed by subsurface environments and inform their
evolutionary history. To phrase this question slightly differently: Is
life in the subsurface sufficiently isolated from that of the surface
to be considered endemic? Demonstrating endemic subsurface
lineages would provide the strongest evidence that the deep
subsurface hosts conditions that are not met in near-surface en-
vironments and remain distinct enough to prohibit the successful
dispersal of subsurface organisms into surface environments or
vice versa. Additional insight into this question will better con-
strain the possibility of life beyond Earth.
Is the Subsurface Microbiome Endemic?
The widespread prevalence of Ca. D. audaxviator (46) among
several fracture waters in South African gold mines (46, 82) pro-
vides the best evidence that life in the subsurface is endemic. A
second example is provided by the Henderson group 1 bacterial
lineage, distantly related to Nitrospirae, that has only been found
(at the species level) in a single >1-km-deep molybdenummine in
Colorado (47). Other Ca. D. audaxviator species-level phylotypes
have only been found in a deep borehole in Nevada (83). Several
studies have identified more distantly related Ca. Desulforudis-
like phylotypes from both deep terrestrial and marine bore-
holes that provide further evidence that the genus is generally
restricted to subsurface environments (77, 84, 85). Regardless of
the precise geographic distribution of Ca. D. audaxviator, their
apparent distribution in discrete subsurface habitats, when con-
sidered with their physiological capacity for self-sustainment in
oligotrophic environments, provide strong evidence that this
taxon is endemic.
However, distributions of other subsurface-typical organisms
into subsurface-only environments have not generally been ob-
served. For instance, the Bathyarchaeota display significant phy-
logenetic diversity and are distributed across a wide range of
environments that include surface sediments, deep-subsurface
environments, and hot springs, among others (72, 73). A recent
analysis of the distribution of Bathyarchaeota 16S rRNA gene
phylotypes across environment types suggested that there are no
phylogenetic subgroups that are uniquely associated with sub-
surface habitats (73). However, several 16S rRNA gene subgroups
were enriched in subsurface habitats, suggesting that there is an
evolutionary signal, if not entirely cohesive, of habitat preference
among Bathyarchaeota phylotypes (73). In contrast, there is no
clear phylogenetic distinction between Aigarchaeota phyotypes
(Ca. Caldiarchaeum subterraneum) that are found in subsurface
environments and those that have been found in surface thermal
features such as hot springs (71). Similarly, although certain 16S
rRNA gene phylogenetic subgroups of Hadesarchaea phylotypes
exhibit enrichment of subsurface-derived sequences, there is not
a strong phylogenetic signal delineating subsurface Hadesarchaea
from those found in surface thermal features, such as in the
Yellowstone National Park hot springs (61).
Available cultivation-independent evidence, compiled over
the past 25 years, suggests that, although there may be some
organisms that are endemic to the deep subsurface (e.g., Ca.
D. audaxviator and Henderson group 1), there is evidence that
many subsurface-typical taxa are also distributed in near-surface
environments, such as hot springs. Indeed, it may be more likely
that the prevailing environmental conditions that characterize
deep, hot, subsurface environments such as oligotrophy, low
nutrient flux, high temperatures, highly reducing conditions,
plentiful lithogenic electron donors (i.e., H2 and CO), and oxidant
limitation select for the same taxa in both near-surface and sub-
surface conditions that exhibit these conditions. Comparisons of
genomes reconstructed from subsurface populations and those
from near-surface populations are needed to further deconvolute
whether specific physiological adaptations differentiate closely
related genotypes that are found in both deep- and near-
surface environments.
What Are the Roles of Hydrocarbons in Sustaining
Subsurface Microbiomes?
A central tenet of Gold’s deep, hot biosphere proposal was the
role of mantle-derived hydrocarbons in supporting microbial life.
Although some laboratory studies have suggested that lighter
hydrocarbons such as ethane, propane, and butane can be gen-
erated today within the Earth’s upper mantle (86, 87), the origin of
most hydrocarbons present in the subsurface environment (e.g., in
Precambrian continental crustal environments) are not likely to be
mantle-derived, as Gold initially proposed (8, 9). However, Gold’s
ideas have led to substantial interest in determining the origin of
subsurface hydrocarbons, and much attention has been directed
toward these efforts. In particular, serpentinization of olivine can
generate elevated H2, and that, when high enough in concen-
tration, may catalyze the production of CH4 (88, 89), although
recent work suggests that the extent of CH4 production may be
contextually dependent on the presence of an H2 steam phase
(90). Regardless of its source, a reservoir of 3 × 1014 m3 of gas
hydrate, primarily in the form of CH4, is thought to exist in the
subsurface (91), providing an abundant source of reductant to
drive subsurface microbial metabolism.
At the time of Gold’s publication, the accepted mechanism for
how microorganisms oxidize CH4 was via aerobic bacterial
methanotrophy (92), which would be of limited importance in
largely anoxic, high-temperature subsurface environments. Al-
though geochemical, isotopic, and activity data (93–95) hinted at
the potential for anaerobic CH4 oxidation at the time of Gold’s
publication, it was not until later that the importance and mech-
anistic details of this process were elucidated. For example, CH4 is
now thought to be oxidized biologically under anoxic conditions
via a variety of means that include a near reversal of the meth-
anogenesis pathway (96, 97) with a variety of metal oxidants (98,
99), nitrite dependent oxidation of methane (100), and the addi-
tion of fumarate to methane (101).
Although the role of CH4 in supporting subsurface life is better
understood, the anaerobic processes involved in the degradation
of hydrocarbons are less well understood. Under oxic conditions,
hydrocarbon degradation is carried out through the activity of
oxygenases and glycyl radical enzymes (102). Comparatively little
is known about high-temperature (>60 °C) oxidation of hydro-
carbons heavier than CH4 by subsurface microorganisms, partic-
ularly under anoxic conditions. However, a thermophilic isolate
from the Guaymas basin hydrocarbon seep has been shown to
grow on propane and n-butane via fumarate addition at 60 °C
(103). More recently, a mechanism for anaerobic butane oxidation
via activation by McrA proteins was demonstrated in thermophilic
archaeal Ca. Syntrophoarchaeum spp. that grow in consortium
with SRB (76). This recent finding represents an exciting new
breakthrough in our understanding of high-temperature anaero-
bic alkane degradation, which could be more widespread than
previously appreciated. Thus, based on currently available data,
the extent to which microorganisms use or modify hydrocarbons
in anoxic, high-temperature environments remains unclear and is
ripe for further study.
The Future
Much of the deep, hot biosphere remains to be discovered, in-
cluding the role of this biota in hydrocarbon production or con-
sumption. Moreover, a number of other outstanding questions
concerning the nature of geobiological controls on subsurface
habitability remain unanswered. For instance, the nature of mi-
croorganisms that mediate water–rock interactions via the con-
sumption or production of reaction products or substrates which
then shape the subsurface geochemical landscape and the
feedbacks that occur between biological and geological pro-
cesses necessitates further study. Additionally, the production of
high-energy substrates such as H2 from mechanical weathering
processes, such as shearing of silica minerals in bedrock during
tectonic activity, has been demonstrated (104), and a similar
process has been suggested to be of key importance in sup-
porting hydrogenotrophs in near-surface environments (25).
Given the prevalence of silicate minerals in Earth’s crust and the
prevalence of tectonic activity both today and in Earth’s past, it is
possible that this mechanism of H2 production plays a role in
supporting a deep, hot biosphere.
The pioneering ideas proposed by Thomas Gold inspired a
generation of researchers in the field of geobiology to dive
deeper into the possibilities of subsurface life, spawning hundreds
of relevant publications (Fig. 1). Importantly, considerable funding
from the National Science Foundation for drilling expeditions over
the previous 10 years, in concert with the Integrated Ocean Dril-
ling Program efforts, have led to tremendous new insights in
marine subsurface settings. Fifteen years ago, the first drilling
expedition that focused on understanding microbial processes in
deeply buried marine sediments was conducted, and an in-
creasing number of expeditions with microbial focus have since
been undertaken, with several more planned. Indeed, without
these funding initiatives, much of the body of research produced
since Gold’s 1992 paper (Fig. 1) would not have been possible.
Nevertheless, a continuing roadblock to further study and
understanding of the deep biosphere is site access, particularly in
terrestrial systems. Boreholes are enormously expensive and dif-
ficult to obtain. The expense associated with access to subsurface
environments and the compelling nature of questions that remain
unanswered require a deep subsurface microbiome initiative that
aims to synthesize research from marine and terrestrial settings.
Such an initiative would provide progress which is greater than
that which can be achieved by a single laboratory or a small group
of laboratories. For example, an entire field exists to drill such
boreholes in the oil and gas industry. If an open collaboration
between scientists and the companies that drill ever deeper to
extract hydrocarbon resources can occur through a deep sub-
surface microbiome initiative, a greater understanding of how
microorganisms interact with the hydrocarbon-rich settings kilo-
meters below our feet will be unveiled. Moreover, an expanse
of underground research laboratories exists for high-energy
physics and radioactive waste disposal, in addition to commer-
cial mines and tunneling projects that have not been studied
microbiologically. Such information obtained from expanded
studies into these subsurface portals will not only allow us to
further our understanding of the limits of life on Earth and address
hypotheses put forth by Gold, it will also allow us to refine our
predictions for the potential for life on other planetary bodies.
Deep hydrocarbon deposits on Mars, Titan, and worlds beyond
could play host to life similar to that in Earth’s own crust. The
techniques used to better study and understand deep, hot bio-
spheres on Earth could then be applied to robotically probe tar-
gets in deep space as we move into the next century of scientific
discovery. Technology is advancing at a rate wherein we may find
that Gold’s deep, hot biosphere is not only true, but common
across the universe. That would be quite a legacy for a man who
routinely thought outside of his own field and thought that he
would have been burned at the stake in a different age.
Acknowledgments
We thank members of the Geo-Environmental Microbiology (GEM) Laboratory
at the Colorado School of Mines and the Geobiology Laboratory at Montana
State University for thoughtful insights, contributions, and reviews. We thank
those who went out on a limb to consider microbial life, and its importance to
the planet, including Drs. David Updegraff, Claude ZoBell, Thomas Gold, Carl
Woese, Ralph Wolfe, Edward Leadbetter, Gerrit Voordouw, Katrina Edwards,
Paul Falkowski, Ralf Conrad, and Abigail Salyers, among many others. The
guidance and mentoring of subsurface science pioneers Dr. Gill Geesey
(Montana State University) and Dr. Norman Pace (University of Colorado
Boulder) has enabled many of us to make meaningful contributions to science.
We also thank Barbara Sherwood Lollar and Jan Amend, whose insights and
suggestions improved this work. This work was supported by NASA Astrobiol-
ogy Institute Grant NNA15BB02A (to E.S.B. and J.R.S.) and by the Zink
Sunnyside Family Fund (J.R.S.).
1 Gold T (1992) The deep, hot biosphere. Proc Natl Acad Sci USA 89:6045–6049.
2 Gold T (1999) The Deep Hot Biosphere (Springer, New York).
3 Ringle K (November 1, 1999) A scientific heretic delves beneath the surface. Washington Post, Section C, p 1.
4 Bondi H (June 28, 2004) Professor Thomas Gold. The Independent, Obituaries Section.
5 Shock EL, Boyd ES (2015) Principles of geobiochemistry. Elements 11:395–401.
6 Thauer RK, Jungermann K, Decker K (1977) Energy conservation in chemotrophic anaerobic bacteria. Bacteriol Rev 41:100–180.
7 Lyons TW, Fike DA, Zerkle A (2015) Emerging biogeochemical views of Earth’s ancient microbial worlds. Elements 11:415–421.
8 Sherwood Lollar B, Westgate TD, Ward JA, Slater GF, Lacrampe-Couloume G (2002) Abiogenic formation of alkanes in the Earth’s crust as a minor source for
global hydrocarbon reservoirs. Nature 416:522–524.
9 Etiope G, Sherwood Lollar B (2013) Abiotic methane on earth. Rev Geophys 51:276–299.
10 Bains W, Xiao Y, Yu C (2015) Prediction of the maximum temperature for life based on the stability of metabolites to decomposition in water. Life (Basel) 5:1054–1100.
11 McCollom TM (1999) Methanogenesis as a potential source of chemical energy for primary biomass production by autotrophic organisms in hydrothermal
systems on Europa. J Geophys Res Planets 104:30729–30742.
12 Onstott TC, et al. (2006) Martian CH4: Sources, flux, and detection. Astrobiology 6:377–395.
13 Nixon SL, Cousins CR, Cockell CS (2013) Plausible microbial metabolisms on Mars. Astron Geophys 54:1.13–1.16.
14 Stevens TO, Mckinley JP (1995) Lithoautotrophic microbial ecosystems in deep basalt aquifers. Science 270:450–454.
15 Chapelle FH, et al. (2002) A hydrogen-based subsurface microbial community dominated by methanogens. Nature 415:312–315.
16 Kral TA, Brink KM, Miller SL, McKay CP (1998) Hydrogen consumption by methanogens on the early Earth. Orig Life Evol Biosph 28:311–319.
17 Schulte M, Blake D, Hoehler T, McCollom T (2006) Serpentinization and its implications for life on the early Earth and Mars. Astrobiology 6:364–376.
18 Boyd ES, Schut GJ, Adams MWW, Peters JW (2014) Hydrogen metabolism and the evolution of biological respiration. Microbe 9:361–367.
19 Freund F, Dickinson JT, Cash M (2002) Hydrogen in rocks: An energy source for deep microbial communities. Astrobiology 2:83–92.
20 Lin LH, et al. (2005) Radiolytic H2 in continental crust: Nuclear power for deep subsurface microbial communities. Geochem Geophys Geosys 6:Q07003.
21 Blair CC, D’Hondt S, Spivack AJ, Kingsley RH (2007) Radiolytic hydrogen and microbial respiration in subsurface sediments. Astrobiology 7:951–970.
22 Sherwood Lollar B, et al. (2007) Hydrogeologic controls on episodic H2 release from precambrian fractured rocks—energy for deep subsurface life on earth and
mars. Astrobiology 7:971–986.
23 Sherwood Lollar B, Onstott TC, Lacrampe-Couloume G, Ballentine CJ (2014) The contribution of the Precambrian continental lithosphere to global H2
production. Nature 516:379–382.
24 Saruwatari K, Kameda J, Tanaka H (2004) Generation of hydrogen ions and hydrogen gas in quartz-water crushing experiments: an example of chemical
processes in active faults. Phys Chem Miner 31:176–182.
25 Telling J, et al. (2015) Rock comminution as a source of hydrogen for subglacial ecosystems. Nat Geosci 8:851–855.
26 Parnell J, Blamey N (2017) Global hydrogen reservoirs in basement and basins. Geochem Trans 18:2.
27 Corliss JB, et al. (1979) Submarine thermal springs on the Galapagos rift. Science 203:1073–1083.
28 Nealson KH (2005) Hydrogen and energy flow as “sensed” by molecular genetics. Proc Natl Acad Sci USA 102:3889–3890.
29 Spear JR, Walker JJ, McCollom TM, Pace NR (2005) Hydrogen and bioenergetics in the Yellowstone geothermal ecosystem. Proc Natl Acad Sci USA
102:2555–2560.
30 Boyd ES, Hamilton TL, Spear JR, Lavin M, Peters JW (2010) [FeFe]-hydrogenase in Yellowstone National Park: evidence for dispersal limitation and phylogenetic
niche conservatism. ISME J 4:1485–1495.
31 Peters JW, et al. (2015) [FeFe]- and [NiFe]-hydrogenase diversity, mechanism, and maturation. Biochim Biophys Acta 1853:1350–1369.
32 Studier MH, Hayatsu R, Anders E (1968) Origin of organic matter in early solar system—I. Hydrocarbons. Geochim Cosmochim Acta 32:175–190.
33 McCollom TM, Seewald JS (2001) A reassessment of the potential for reduction of dissolved CO2 to hydrocarbons during serpentinization of olivine. Geochim
Cosmochim Acta 65:3769–3778.
34 Helz GR, et al. (1996) Mechanism of molybdenum removal from the sea and its concentration in black shales: EXAFS evidence. Geochim Cosmochim Acta
60:3631–3642.
35 Russell MJ, Martin W (2004) The rocky roots of the acetyl-CoA pathway. Trends Biochem Sci 29:358–363.
36 Boyd ES, et al. (2014) Origin and evolution of Fe-S proteins and enzymes. Iron-Sulfur Clusters in Chemistry and Biology, ed Rouault T (De Gruyter, Berlin), pp
619–636.
37 Kurr M, et al. (1991) Methanopyrus kandleri, gen. and sp. nov. represents a novel group of hyperthermophilic methanogens, growing at 110°C. Arch Microbiol
156:239–247.
38 Takai K, et al. (2008) Cell proliferation at 122 ° C and isotopically heavy CH4 production by a hyperthermophilic methanogen under high-pressure cultivation.
Proc Natl Acad Sci USA 105:10949–10954.
39 Zobell CE (1947) Microbial transformation of molecular hydrogen in marine sediments, with particular reference to petroleum. AAPG Bull 31:1709–1751.
40 Chapelle FH, Zelibor JL, Grimes DJ, Knobel LL (1987) Bacteria in deep coastal-plain sediments of Maryland—a possible source of CO2 to groundwater. Water
Resour Res 23:1625–1632.
41 Pedersen K, Ekendahl S (1990) Distribution and activity of bacteria in deep granitic groundwaters of southeastern Sweden. Microb Ecol 20:37–52.
42 Stetter KO, et al. (1993) Hyperthermophilic archaea are thriving in deep North Sea and Alaskan oil reservoirs. Nature 365:743–745.
43 Takai K, Moser DP, DeFlaun M, Onstott TC, Fredrickson JK (2001) Archaeal diversity in waters from deep South African gold mines. Appl Environ Microbiol
67:5750–5760.
44 Cowen JP, et al. (2003) Fluids from aging ocean crust that support microbial life. Science 299:120–123.
45 Nunoura T, et al. (2005) Genetic and functional properties of uncultivated thermophilic crenarchaeotes from a subsurface gold mine as revealed by analysis of
genome fragments. Environ Microbiol 7:1967–1984.
46 Chivian D, et al. (2008) Environmental genomics reveals a single-species ecosystem deep within Earth. Science 322:275–278.
47 Sahl JW, et al. (2008) Subsurface microbial diversity in deep-granitic-fracture water in Colorado. Appl Environ Microbiol 74:143–152.
48 Orcutt BN, et al. (2011) Colonization of subsurface microbial observatories deployed in young ocean crust. ISME J 5:692–703.
49 Hug LA, et al. (2016) A new view of the tree of life. Nat Microbiol 1:16048.
50 Edwards KJ, Becker K, Colwell F (2012) The deep, dark energy biosphere: Intraterrestrial life on Earth. Annu Rev Earth Planet Sci 40:551–568.
51 Froehlich PN, et al. (1979) Early oxidation of organic matter in pelagic sediments of the eastern equatorial Atlantic: Suboxic diagenesis. Geochim Cosmochim
Acta 43:1075–1090.
52 D’Hondt S, Rutherford S, Spivack AJ (2002) Metabolic activity of subsurface life in deep-sea sediments. Science 295:2067–2070.
53 LaRowe DE, Burwicz E, Arndt S, Dale AW, Amend JP (2017) Temperature and volume of global marine sediments. Geology 45:275–278.
54 Onstott TC, et al. (1999) A global perspective on the microbial abundance and activity in the deep subsurface. Enigmatic Microorganisms and Life in Extreme
Environments, ed Seckbach J (Springer, Dordrecht), Vol 1, pp 487–500.
55 Ward JA, et al. (2004) Microbial hydrocarbon gases in the Witwatersrand Basin, South Africa: Implications for the deep biosphere. Geochim Cosmochim Acta
68:3239–3250.
56 Hara K, et al. (2005) Analysis of the archaeal sub-seafloor community at Suiyo Seamount on the Izu-Bonin Arc. Adv Space Res 35:1634–1642.
57 Huber JA, Johnson HP, Butterfield DA, Baross JA (2006) Microbial life in ridge flank crustal fluids. Environ Microbiol 8:88–99.
58 Roussel EG, et al. (2008) Extending the sub-sea-floor biosphere. Science 320:1046.
59 Teske A, Sørensen KB (2008) Uncultured archaea in deep marine subsurface sediments: Have we caught them all? ISME J 2:3–18.
60 Nunoura T, et al. (2011) Insights into the evolution of Archaea and eukaryotic protein modifier systems revealed by the genome of a novel archaeal group.
Nucleic Acids Res 39:3204–3223.
61 Baker BJ, et al. (2016) Genomic inference of the metabolism of cosmopolitan subsurface Archaea, Hadesarchaea. Nat Microbiol 1:16002.
62 Moser DP, et al. (2005) Desulfotomaculum and Methanobacterium spp. dominate a 4- to 5-kilometer-deep fault. Appl Environ Microbiol 71:8773–8783.
63 Takai K, et al. (2002) Isolation and metabolic characteristics of previously uncultured members of the order aquificales in a subsurface gold mine. Appl Environ
Microbiol 68:3046–3054.
64 Hirayama H, et al. (2005) Bacterial community shift along a subsurface geothermal water stream in a Japanese gold mine. Extremophiles 9:169–184.
65 Takami H, et al. (2012) A deeply branching thermophilic bacterium with an ancient acetyl-CoA pathway dominates a subsurface ecosystem. PLoS One 7:e30559.
66 Labonte´ JM, et al. (2015) Single cell genomics indicates horizontal gene transfer and viral infections in a deep subsurface Firmicutes population. Front Microbiol
6:1–11.
67 Liu Y, Whitman WB (2008) Metabolic, phylogenetic, and ecological diversity of the methanogenic archaea. Ann N Y Acad Sci 1125:171–189.
68 LauMC, et al. (2016) An oligotrophic deep-subsurface community dependent on syntrophy is dominated by sulfur-driven autotrophic denitrifiers. Proc Natl Acad
Sci USA 113:E7927–E7936.
69 Pereira IA, et al. (2011) A comparative genomic analysis of energy metabolism in sulfate reducing bacteria and archaea. Front Microbiol 2:1–22.
70 Beam JP, et al. (2016) Ecophysiology of an uncultivated lineage of Aigarchaeota from an oxic, hot spring filamentous ‘streamer’ community. ISME J 10:210–224.
71 Hedlund BP, et al. (2015) Uncultivated thermophiles: current status and spotlight on ‘Aigarchaeota’ Curr Opin Microbiol 25:136–145.
72 Teske AP (2006) Microbial communities of deep marine subsurface sediments: Molecular and cultivation surveys. Geomicrobiol J 23:357–368.
73 Lazar CS, et al. (2015) Environmental controls on intragroup diversity of the uncultured benthic archaea of the miscellaneous Crenarchaeotal group lineage
naturally enriched in anoxic sediments of the White Oak River estuary (North Carolina, USA). Environ Microbiol 17:2228–2238.
74 Evans PN, et al. (2015) Methane metabolism in the archaeal phylum Bathyarchaeota revealed by genome-centric metagenomics. Science 350:434–438.
75 He Y, et al. (2016) Genomic and enzymatic evidence for acetogenesis among multiple lineages of the archaeal phylum Bathyarchaeota widespread in marine
sediments. Nat Microbiol 1:16035.
76 Laso-Pe´rez R, et al. (2016) Thermophilic archaea activate butane via alkyl-coenzyme M formation. Nature 539:396–401.
77 Lin LH, et al. (2006) Long-term sustainability of a high-energy, low-diversity crustal biome. Science 314:479–482.
78 Lever MA, et al. (2013) Evidence for microbial carbon and sulfur cycling in deeply buried ridge flank basalt. Science 339:1305–1308.
79 Jungbluth SP, Grote J, Lin HT, Cowen JP, Rappe´ MS (2013) Microbial diversity within basement fluids of the sediment-buried Juan de Fuca Ridge flank. ISME J
7:161–172.
80 Jungbluth SP, Lin HT, Cowen JP, Glazer BT, Rappe´ MS (2014) Phylogenetic diversity of microorganisms in subseafloor crustal fluids from Holes 1025C and 1026B
along the Juan de Fuca Ridge flank. Front Microbiol 5:119.
81 Jungbluth SP, Amend JP, Rappe´ MS (2017) Metagenome sequencing and 98 microbial genomes from Juan de Fuca Ridge flank subsurface fluids. Sci Data
4:170037.
82 Magnabosco C, et al. (2014) Comparisons of the composition and biogeographic distribution of the bacterial communities occupying South African thermal
springs with those inhabiting deep subsurface fracture water. Front Microbiol 5:1–17.
83 Moser DP (2012) Deep microbial ecosystems in the U.S. Great Basin: A second home for Desulforudis audaxviator? 2012 American Geophysical Union Fall
Meeting, December 3–7, San Francisco. Available at abstractsearch.agu.org/meetings/2012/FM/B41F-08.html. Accessed May 23, 2017.
84 Itävaara M, et al. (2011) Characterization of bacterial diversity to a depth of 1500 m in the Outokumpu deep borehole, Fennoscandian Shield. FEMS Microbiol
Ecol 77:295–309.
85 Tiago I, Verı´ssimo A (2013) Microbial and functional diversity of a subterrestrial high pH groundwater associated to serpentinization. Environ Microbiol
15:1687–1706.
86 Kolesnikov A, Kutcherov VG, Goncharov AF (2009) Methane-derived hydrocarbons produced under upper-mantle conditions. Nat Geosci 2:566–570.
87 Scott HP, et al. (2004) Generation of methane in the Earth’s mantle: In situ high pressure-temperature measurements of carbonate reduction. Proc Natl Acad Sci
USA 101:14023–14026.
88 Barnes I, Lamarche VC, Jr, Himmelberg G (1967) Geochemical evidence of present-day serpentinization. Science 156:830–832.
89 Abrajano TA, Sturchio NC (1990) Geochemistry of reduced gas related to serpentinization of the Zambales ophiolite, Philippines. Appl Geochem 5:625–630.
90 McCollom TM (2016) Abiotic methane formation during experimental serpentinization of olivine. Proc Natl Acad Sci USA 113:13965–13970.
91 Boswell R, Collett TS (2011) Current perspectives on gas hydrate resources. Energy Environ Sci 4:1206–1215.
92 Hanson RS, Hanson TE (1996) Methanotrophic bacteria. Microbiol Rev 60:439–471.
93 Martens CS, Berner RA (1977) Interstitial water chemistry of anoxic Long Island Sound sediments. 1. Dissolved-gases. Limnol Oceanogr 22:10–25.
94 Iversen N, Jorgensen BB (1985) Anaerobic methane oxidation rates at the sulfate methane transition in marine-sediments from Kattegat and Skagerrak
(Denmark). Limnol Oceanogr 30:944–955.
95 Alperin MJ, Reeburgh WS, Whiticar MJ (1988) Carbon and hydrogen isotope fractionation resulting from anaerobic methane oxidation. Global Biogeochem
Cycles 2:279–288.
96 Scheller S, Goenrich M, Boecher R, Thauer RK, Jaun B (2010) The key nickel enzyme of methanogenesis catalyses the anaerobic oxidation of methane. Nature
465:606–608.
97 Scheller S, Goenrich M, Mayr S, Thauer RK, Jaun B (2010) Intermediates in the catalytic cycle of methyl coenzyme M reductase: Isotope exchange is consistent
with formation of a σ-alkane-nickel complex. Angew Chem Int Ed Engl 49:8112–8115.
98 Beal EJ, House CH, Orphan VJ (2009) Manganese- and iron-dependent marine methane oxidation. Science 325:184–187.
99 Lai CY, et al. (2016) Selenate and nitrate bioreductions using methane as the electron donor in a membrane biofilm reactor. Environ Sci Technol 50:10179–10186.
100 Ettwig KF, et al. (2010) Nitrite-driven anaerobic methane oxidation by oxygenic bacteria. Nature 464:543–548.
101 Beasley KK, Nanny MA (2012) Potential energy surface for anaerobic oxidation of methane via fumarate addition. Environ Sci Technol 46:8244–8252.
102 Wang W, Shao Z (2013) Enzymes and genes involved in aerobic alkane degradation. Front Microbiol 4:1–7.
103 Kniemeyer O, et al. (2007) Anaerobic oxidation of short-chain hydrocarbons by marine sulphate-reducing bacteria. Nature 449:898–901.
104 Sugisaki R, et al. (1983) Origin of hydrogen and carbon dioxide in fault gases and its relation to fault activity. J Geol 91:239–258.