Article
https://doi.org/10.1038/s41467-024-53438-4
Isotopic evidence of acetate turnover in
Precambrian continental fracture
fl
uids
Elliott P. Mueller
1
, Juliann Panehal
1
, Alexander Meshoulam
1
, Min Song
2
,
Christian T. Hansen
3
,OliverWarr
2,4
, Jason Boettger
5
, Verena B. Heuer
3
,
Wolfgang Bach
3
, Kai-Uwe Hinrichs
3
, John M. Eiler
1
, Victoria Orphan
1
,
Barbara Sherwood Lollar
2,6
& Alex L. Sessions
1
The deep continental crust represents a va
st potential habitat for microbial life
where its activity remains poorly const
rained. Organic acids like acetate are
common in these ecosystems, but their
role in the subsurface carbon cycle -
including the mechanism and rate of their turnover - is still unclear. Here, we
develop an isotope-exchange
‘
clock
’
based on the abiotic equilibration of
H-isotopes between acetate and water, which can be used to de
fi
ne the max-
imum in situ acetate residence time. W
e apply this technique to the fracture
fl
uids in Birchtree and Kidd Creek min
es within the Canadian Precambrian
crust. At both sites, we
fi
nd that acetate residence times are <1 million years
and calculated a rate of turnover that co
uld theoretically support microbial
life. However, radiolytic water-rock re
actions could also contribute to acetate
production and degradation, a proces
s that would have global relevance for
thedeepbiosphere.Morebroadly,our
study demonstrates the utility of
isotope-exchange clocks in determining residence times of biomolecules with
possible applications to other environments.
Fluid-bearing fractures within crystalline rocks of the Precambrian
continental crust have been identi
fi
ed globally at sites from the
Canadian Shield to the South African Craton and may store as much
as one-third of the Earth
’
s groundwater
1
. Surface meteoric water
mixes with fracture
fl
uids in the top 1
–
2 kilometers of the crust
sustaining diverse populations of microorganisms. Here, we focus
on still deeper
fl
uids that are generally characterized by anoxia, high
salinities (up to 325 g/L), low cell densities (<10
3
–
10
5
cells/L) and
variable hydrogeologic recharge rates
2
–
4
. At the Kidd Creek Cu-Zn-
Ag Mine (Timmins, Ontario), noble gas-derived mean residence
times of fracture
fl
uids can exceed 10
9
years
3
. Long
fl
uid residence
times allow the products of water-rock reactions to accumulate to a
greater extent than elsewhere. Despite the accumulation of these
potential substrates, cell densities in the
fl
uids are low, making the
Kidd Creek Deep Fluid and Deep Life Observatory a prime window
into abiogenic synthesis
4
. Most notably, radiolysis produces abun-
dant H
2
while simultaneously generating oxidants like sulfate
5
–
9
.At
suf
fi
ciently high concentrations, H
2
can reduce inorganic carbon to
generate methane and higher hydrocarbons through abiotic Saba-
tier and polymerization reactions
10
–
13
. It was recently suggested,
based on laboratory experiments, that radiolysis in Kidd Creek may
also generate simple organic acids such as acetate, formate and
oxalate from water and dissolved inorganic carbon
14
–
16
. Indeed, the
dissolved organic carbon pool in Kidd Creek
’
s fracture waters is
over 2 mM and up to 68% of this pool is composed solely of acetate
and formate
16
. Through observations of Kidd Creek and other sub-
surface continental sites, it has become clear that abiotic water-rock
reactions including radiolysis can provide a chemical framework
–
organic carbon, oxidants and reductants
–
that could support
microbial communities
17
.
Received: 30 December 2023
Accepted: 8 October 2024
Check for updates
1
Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, CA, USA.
2
Department of Earth Sciences, University of Toronto,
Toronto, ON, Canada.
3
MARUM Centre for Marine Environmental Sciences, University of Bremen, Bremen, Germany.
4
Department of Earth Sciences,
University of Ottawa, Ottawa, ON, Canada.
5
Department of Earth, Environmental, and Resource Sciences, University of Texas at El Paso, El Paso, TX, USA.
6
Institut de Physique du Globe de Paris (IPGP), Université Paris Cité, 1 rue Jussieu, Paris, France.
e-mail:
elliottpmueller@gmail.com
Nature Communications
| (2024) 15:9130
1
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1234567890():,;
The synthesis mechanism of these chemical species has been
studied for over thirty years at Kidd Creek, yet estimates of their
turnover times are to date limited. Methane and sulfur cycling have
been examined through isotopic analyses, but these measurements
provide binary statements about production and consumption rather
than quantitative rates
10
,
18
. Substrate turnover times are instead esti-
mated via bottom-up models of radiolytic yields that come with large
uncertainties
5
–
7
,
9
. Direct measurements of carbon turnover are needed
for accurate evaluation of the net productivity and thus habitability of
hydrogeologically isolated systems like Kidd Creek. Moreover, envir-
onmental measurements of abiogenesis rates could elucidate the
quantitative importance of these reactions in other deep biosphere
locations both on Earth and potentially other planets or moons.
Here, we constrain the turnover time of acetate in two deep
subsurface fracture
fl
uid systems by developing and applying an
isotope-exchange clock for dissolved acetate. First, we experimentally
constrained the rate of uncatalyzed (abiotic) H-isotope exchange
between water and acetate methyl-H, which is presumed to occur
through a tautomerization reaction
19
,
20
.Wefoundthattherateofthis
exchange reaction follows a
fi
rst-order Arrhenius relationship with
temperature (Fig.
1
A). Since acetate is synthesized out of H-isotopic
equilibrium with surrounding
fl
uids and exchange drives it towards
equilibrium at a known rate, the apparent
2
H-fractionation between
acetate and water can serve as a clock: If acetate turnover is slower
than abiotic isotopic exchange, acetate
’
smethyl-site
δ
2
Hcomposition
will be de
fi
ned by the water
δ
2
H and the equilibrium isotope effect (EIE)
between them. Alternatively, if turnover is comparatively high, it will
have a disequilibrated signature from the water. Although we do not
(yet) know the magnitude of starting disequilibrium upon acetate
synthesis, preventing a fully quantitative estimate of residence time,
the mere presence of isotopic disequilibrium between acetate and
water must indicate a residence time that is shorter than the equili-
bration time.
We applied this approach to fracture
fl
uids at Kidd Creek Mine
and
–
for comparison
–
at Birchtree Mine, a site with lower salinity and
higher microbial activity in the Canadian Shield
16
. A suite of microbial
communities with diverse metabolisms have been enriched from
fl
uids
from Thompson Mine, adjacent to the Birchtree site, including fer-
mentation and organoclastic sulfate reduction
21
. Whereas only alkane-
oxidizing and hydrogenotrophic sulfate reducers could be enriched
from Kidd Creek
fl
uids
4
. Cell densities are also higher in Thompson
fl
uids (10
3
−
10
7
cells/mL) than in Kidd Creek (<10
4
cells/mL)
4
,
21
.The
distinct carbon isotope ratios of acetate in Birchtree (
−
27
‰
)andKidd
Creek (
−
7
‰
)
fl
uids further supported the hypothesis that microbial
communities were actively turning over dissolved organic molecules
like acetate in Birchtree
fl
uids, while Kidd Creek
fl
uids represented an
abiotic endmember with long organic residence times
16
.Weusedour
isotope exchange clock method to test this hypothesis and found
acetate-water
2
H disequilibria at Birchtree that con
fi
rm acetate turn
over, likely by microbial metabolisms. More notably, acetate-water
disequilibria was also identi
fi
ed in Kidd Creek
fl
uid, indicating rela-
tively short acetate residence times (<1 Myr) despite
fl
uid residence
times that are 1000-times longer. Our results from Kidd Creek provide
insights into an active carbon cycle within isolated deep continental
fracture
fl
uids and suggest tentative constraints on the importance of
radiolytic acetate production as an abiotic reaction in the deep
biosphere.
Results and discussion
Experimental rates of hydrogen isotope exchange between
acetate methyl hydrogen and water
Acetate was incubated at temperatures between 60 °C and 200 °C in
the presence of 5% deuterated water in pressurized gold bags (see
Methods). To derive the kinetic rate constant for hydrogen exchange
between acetate
’
s methyl group and ambient water, the
2
H/
1
Hratio
(
δ
2
H value) of acetate
’
s methyl group was measured periodically
throughout the incubations via ESI-Orbitrap mass spectrometry (See
Methods)
22
. Under every condition tested, acetate
δ
2
Hvalues
increased with time re
fl
ecting exchange with the
2
H-enriched aqueous
medium. At high temperatures (
≥
150 °C), the rate of acetate
2
H
enrichment over time was initially linear then gradually
fl
attened as it
approached isotopic equilibrium with water (Fig. S2). At lower tem-
peratures, the exchange kinetics were too slow to allow full equili-
bration of acetate and water within the runtime of the experiments.
The
fi
tted half-times for exchange increased exponentially with
decreasing temperature from 3 hours to 810 years, following an
Arrhenius relationship (
R
2
= 0.999,
E
A
= 138 kJ/mol, Fig.
1
). Replicate
incubations, which were performed for all conditions except 100 °C,
resulted in similar reaction rates (overlapping data points in Fig.
1
,
Table S3). Exchange between acetate
’
s methyl-site and water is pre-
sumed to occur through a reversible tautomerization between
ethanoate and ethenol moieties (Fig. S4). Regardless of the exact
Fig. 1 | Acetate exchanges hydrogen isotopes with water at a temperature-
dependent rate. A
Arrhenius plot of hydrogen isotope exchange rates with a linear
regression through experiments at 60 °C (
n
=3),100°C(
n
=1),150°C(
n
=2)and
200 °C (
n
= 2) (solid circles). Extrapolated reaction rates are projected to 25 °C
(open circle). Shaded region represents 2 RMSD.
B
Carbon and hydrogen isotope
composition of acetate from Kidd Creek and Birchtree mines. Shaded regions
represent
δ
13
C of total organic carbon from the metasedimentary rocks of the Kidd
Creek formation
34
. Error bars re
fl
ect standard deviation on analytical triplicates.
Article
https://doi.org/10.1038/s41467-024-53438-4
Nature Communications
| (2024) 15:9130
2
mechanism, the excellent
fi
t to an Arrhenius relationship between
60 °C and 200 °C suggests that the mechanism of exchange does not
change within the tested temperature range. Extrapolating to the
ambient temperature for samples collected at Kidd Creek and Birch-
tree (25 °C), the predicted exchange half-time was 250,000 ± 70,000
years (2xRMSD).
Determining the equilibrium isotope effect
Equilibrium
2
H-isotope effects (EIEs) for acetate-water were calculated
using density functional theory (DFT) across a range of temperatures
(see Methods). These indicated a temperature-dependent change in
the EIE from
−
108
‰
at 250 °C to
−
192
‰
at 25 °C (Fig.
2
B). Four high
temperature incubations at 200 °C were designed to experimentally
test these calculations. Incubations were started with varying magni-
tudes and direction of isotopic disequilibrium, but in each case acetate
δ
2
H values changed with time until the experiments converged to
similar EIEs. Water was present in excess and so did not change in
δ
2
H
value. Equilibrium was reached in less than one day at 200 °C and
remained there for two days (Fig.
2
A). On average, the measured EIE
(0.888 ± 0.012) was within analytical error of the DFT-calculated value
(0.882). While the two experimental series did not perfectly converge
in
δ
2
H values, they came within ~20
‰
of each other. This offset is
potentially due to analytical artifacts associated with measuring the
high
δ
2
H value of acetate in the
2
H
2
O spiked sample and is small in
comparison to the scale of natural hydrogen isotope variations (blue,
Fig.
2
A). Thus, at 200 °C, the empirically determined EIE corroborates
the DFT calculations.
Carbon and hydrogen isotope compositions of acetate from
deep mines
The
δ
13
Cand
δ
2
H values of acetate extracted from Kidd Creek and
Birchtree fracture
fl
uids were measured via the ESI-Orbitrap method,
revealing different isotopic compositions at the two sites
22
.Samples
collected from three separate boreholes in Kidd Creek between 2008
and 2018 yielded
δ
13
Cvaluesof
−
10.0
‰
to
−
6.6
‰
(VPDB) and
δ
2
H
values of
−
142
‰
to
−
130
‰
(VSMOW). In contrast, acetate extracted
from three fracture
fl
uid samples from Birchtree yielded
δ
13
C values of
-26.7
‰
to
−
27.4
‰
and
δ
2
H values of
−
167
‰
to
−
170
‰
(Fig.
2
and
Table S2). All
δ
13
C values match the range of values previously reported
for these two sites
16
. When compared to the previously-measured
δ
2
H
values of water from Kidd Creek and Birchtree (
−
36
‰
and
−
74
‰
,
respectively)
19
, a similar apparent hydrogen isotope fractionation
between acetate and water exists at both sites. This fractionation
ranges from
−
115
‰
to
−
90
‰
(Fig.
2
B) and differs from isotopic equi-
librium at 25 °C by over 50
‰
. These data demonstrate that acetate in
Kidd Creek and Birchtree fracture
fl
uids is far from the calculated
H-isotopic equilibrium with water and must therefore have rates of
production and consumption that are faster than the rate of abiotic
exchange.
The identical apparent acetate-water hydrogen isotope effect
(
2
ε
acetate/water
) from the two sites is notable (Fig.
2
B). One possibility
that we considered is whether complexation of acetate by the abun-
dant (>1 M) dissolved cations
4
could signi
fi
cantly alter the EIE, i.e. a
‘
matrix effect
’
. In this case, a shared
2
ε
acetate/water
value between the
sites would be possible if acetate at both sites was in equilibrium with
water and the
2
ε
acetate/water
valuematchedtheshiftedEIE.Calciumis
the most abundant cation in Kidd Creek and Birchtree
fl
uids that
complexes with free acetate, thus the Ca-acetate complex represents
the most likely acetate complexation in these systems. To test whether
complexation shifts the calculated EIE, we calculated the partition
function ratio of a calcium-acetate bidentate complex and for high
ionic strength brines then combined these to de
fi
ne an EIE for the
complex-brine equilibrium. Conservatively assuming that all the acet-
ate is ligated to calcium cations and is in equilibrium with a CaCl
2
brine,
the calculated EIE is
−
167
‰
at 25 °C, which is 60
‰
offset from the
fractionation observed in Kidd Creek and Birchtree (Fig.
2
B). Thus, a
comparison of DFT calculations and environmental data suggest that
acetate and water in Kidd Creek and Birchtree are in substantial iso-
topic disequilibrium, whether acetate exists as a free anion or is
complexed to calcium in solution. The identical value of
2
ε
acetate/water
values observed at both sites (Fig.
2
B) may instead re
fl
ect kinetic iso-
tope effects that provide insight into acetate turnover mechanisms.
Acetate is cycled in the continental deep subsurface
The turnover times of organic molecules can provide important con-
straints on the productivity and habitability of isolated systems like the
continental deep biosphere, but to date such timescales have been
dif
fi
cult to measure
17
. Water-rock reactions in
fl
uencing the geochem-
istry of Kidd Creek and other sites often operate too slowly to replicate
through experimentation. Similarly, microbial growth rates and
metabolic
fl
uxes typical of these settings are inaccessibly slow on
laboratory timescales
2
. While these processes can be identi
fi
ed
through isotope geochemistry and genomic analyses, rates of abio-
genesis and/or microbial metabolism remain elusive
23
. Our new
Fig. 2 | Theoretical calculations and empirical results con
fi
rm that acetate in
subsurface fracture
fl
uids is in isotopic disequilibrium with the ambient water.
A
Observed isotope effect between acetate and water throughout a three-day
200 °C exchange experiment with water at either
−
50
‰
or +110
‰
. Dashed line
represents the calculated EIE between acetate and pure water. Error bars represent
standard deviation on analytical replicates (
n
=3).
B
Hydrogen isotope
fractionation between acetate and water (
2
ε
acetate/water
) at both sites. Solid line is the
calculated EIE between the Ca-acetate complex and brine water. Dashed line is the
EIE between free acetate and brine water as a function of temperature. Error bars
are covered by the data points and represent standard deviation on analytical
replicates (
n
=3).
Article
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Nature Communications
| (2024) 15:9130
3
H-isotope exchange clock helps to
fi
ll that gap by setting upper limits
on residence times (i.e. lower limits on production and consumption
rates) for acetate. Moreover, the general approach should be directly
applicable to other organic molecules in the environment
including many potentially important organic substrates and
biomolecules.
In fracture
fl
uids from both Kidd Creek and Birchtree, isotopic
disequilibrium between acetate and water implies active production
and consumption of acetate by physical, chemical, and/or biological
processes. These processes must generate and consume acetate faster
than the abiotic exchange reaction can establish H-isotope equilibrium
with water. Given that equilibration of hydrogen atoms occurs in less
than four half-times, acetate residence times must be less than one
million years, at least 1000-fold shorter than that of Kidd Creek frac-
ture
fl
uids (>1 Gyr). Normalizing by the concentrations of acetate
(Table S2) and assuming present-day concentrations are at steady-
state, these turnover times require acetate production and consump-
tion rates of >1 nM/year and >0.1 nM/year in Kidd Creek and Birchtree,
respectively. Since estimated physical
fl
uid recharge rates are slower
than acetate turnover times
3
, our data suggest active production and
consumption of acetate by microbial metabolisms and/or abiotic
reactions.
Acetate consumption could support microbial communities
Many anaerobic microorganisms use acetate as a carbon and electron
source. The rates of acetate consumption implied by our residence
time estimates provide an opportunity to quantify the amount of
metabolic power potentially available to microbes consuming this
substrate in the continental deep biosphere. Anaerobic respiration
–
represented here as sulfate reduction
–
and methanogenesis are
common acetate consumption pathways in anoxic environments
24
,
25
.
Considering the lower threshold of 1 nM/year for acetate consumption
in Kidd Creek, acetate would supply 10
−
11.5
W/L or 10
−
12
W/L via sulfate
reduction or methanogenesis, respectively (Fig.
3
). Assuming a range
of cell-speci
fi
c maintenance powers (the
fl
ux of energy required to
maintain a cell)
26
–
28
,thisratecouldsupportbetween10
2
to 10
6
cells/mL
(Fig.
3
). In saline fracture
fl
uids of the continental subsurface, microbial
cells must synthesize organic osmolytes to combat high osmotic
pressures, increasing their basal power demands
29
,
30
.Ourresultssug-
gest that even with these higher power requirements, at least 10
3
cells/
mL could theoretically survive solely on acetotrophic metabolic
pathways in Kidd Creek (Fig.
3
). However, such calculations only reveal
the viability of these prospective metabolic pathways and cannot be
used as sole evidence of microbial acetotrophy. Further evidence is
required to determine whether acetate is actively being consumed by
biotic processes.
Constraining acetate sources and sinks in the subsurface
The processes producing and degrading acetate can be constrained
using its steady-state isotopic composition. In anoxic settings, acetate
typically has a
δ
13
C value similar to that of the surrounding total
organic carbon (TOC). This is commonly attributed to minimal isotope
effects associated with the production of acetate by microbial fer-
mentation and consumption by anaerobic respiration
31
–
33
. Acetate in
Birchtree fracture
fl
uids has
δ
13
C values that match this expectation,
but it does not have the characteristic
13
Cand
2
H depletion associated
with chemolithoautotrophic acetogenesis
22
,
33
–
35
. This suggests that
acetate turnover in Birchtree
fl
uids is driven by heterotrophic micro-
bial metabolisms.
In contrast, acetate in Kidd Creek is
13
C-enriched relative to TOC
36
.
If microbial activity is similarly responsible for acetate turnover in Kidd
Creek fracture
fl
uids, the reactions(s) consuming acetate must have
larger (normal) carbon isotope effects than those in Birchtree. Acet-
oclastic methanogenesis exhibits such an isotope effect (25-30
‰
)
37
.
When the fermentation of organic matter to acetate is coupled with
methanogenic consumption, acetate can indeed be
13
C-enriched rela-
tive to TOC; however, this enrichment is not consistent across envir-
onments and the mechanisms behind it are still unclear
25
,
32
,
38
,
39
.
Furthermore, the isotopic composition of methane and low ratio of
methane-to-higher-alkanes in Kidd Creek
fl
uids are not consistent with
the signi
fi
cant rates of acetoclastic methanogenesis required to gen-
erate the observed
13
C enrichment in acetate
11
,
37
.Duringcultivation
studies, autotrophic and alkane-oxidizing sulfate reducers were enri-
ched from Kidd Creek
fl
uids, but fermentative and acetoclastic
methanogenic microorganisms were not
4
. Importantly, the lack of
microbial growth does not preclude these metabolic niches from
being an important component o
f the ecosystem. When culture-
independent 16S rRNA sequencing was performed on the same bore-
hole
fl
uids, a variety of putatively chemolithoautotrophic and organ-
isms were identi
fi
ed, including
Fuchsiella ferrireducens
,aniron-
reducing bacterium capable of reductive acetogenesis
40
. While acet-
ogenesis is a possible source of acetate in these systems, cultured
acetogens consistently generate
13
Cand
2
H depleted acetate, the
opposite signal to what is observed here in Kidd Creek
fl
uids
22
,
31
–
35
.
Fig. 3 | Acetate cycling could theoretically support microbial communities in the continental subsurface.
Theoretical cell densities for sulfate reducers (left) and
acetoclastic methanogens (right) that could be supported in the fracture
fl
uids over a range of acetate production rates.
Article
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Nature Communications
| (2024) 15:9130
4
As such, other mechanisms should be considered to explain acetate
turnover in this system.
The identical hydrogen isotope fractionations between acetate
and water at Kidd Creek and Birchtree could indicate turnover
mechanisms that are shared between the mines, such as radiolytic
reactions. Radiolysis is well documented in the deep biosphere and has
been shown to both produce and degrade acetate in laboratory
experiments
14
,
15
. Radiolytic reactions occur when alpha, beta and
gamma irradiation from natural decay of U, Th and K in the rock matrix
triggers reactions with surrounding water, solutes, and minerals
8
,
35
.
Since radiolysis drives substantial abiotic chemistry in subsurface
fl
uids (i.e. H
2
production
5
,
7
), it could produce acetate in situ as well
14
–
16
.
If radiolytic synthesis is the source of acetate in these fracture
fl
uids, it operates at a rate that far e
xceeds those observed in labora-
tory studies. Maximum net yield during in vitro experiments is 6 nM
acetate per joule of alpha radiation, corresponding to 0.007 nM/yr
acetate generation rate in Kidd Creek
fl
uids (see Methods), well below
the minimum production rate estimated here
15
. These results should
be interpreted with caution though. Radiolytic synthesis of organic
acids is not a single production reaction but a network of reactions that
both creates and degrades acetate
14
,
15
. The net yield measured in vitro
represents a balance of production and degradation
fl
uxes, whereas
gross yields could be much higher. Thus, if radiolysis is both producing
and degrading acetate in situ, it could support fast turnover times
without having high net generation rates. Kinetic isotope effects
associated with this turnover could then explain the constant hydro-
gen isotope fractionation from water observed at both sites. However,
while radiolysis is likely cycling acetate in the continental subsurface to
some extent, we cannot presently determine whether it is
solely
responsible for acetate turnover based on our current understanding.
Future work should carefully examine radiolytic reactions under
conditions that match the subsurface to assess their rates of acetate
turnover and associated isotope effects. Given that the substrates for
radiolysis
–
water and DIC
–
are ubiquitous, this process could provide
a means to fuel acetotrophic metabolisms in environments well
beyond the Precambrian continental subsurface, including global
marine sediments, groundwaters, and the subsurface of other planets
or moons.
Isotope-exchange clocks may have wide-ranging applications
Isotope-exchange clocks may also be relevant for other molecules and
environments. For isolated systems characterized by slow turnover
(i.e. subsurface environments of Earth, Mars or Europa), the acetate
H-exchange reaction introduced here could be a useful constraint on
acetate residence times or could simply con
fi
rm the presence of an
active carbon cycle. However, for more biologically productive envir-
onments with fast turnover of organics (i.e. shallow marine
sediments
41
), this particular clock is insensitive. Isotope exchange in
organic molecules that experience more rapid equilibration of
C-bound H would provide more useful information about substrate
turnover in these systems. Molecules containing acidic alpha-H atoms,
which can undergo tautomerization more easily than acetate (i.e.
longer chain organic acids and aldehydes), are potential targets
42
.
Conversely, molecules with yet slower exchange (e.g. alkanes) could
provide information about turnover in hotter environments
43
.Our
study provides the analytical and experimental basis for developing
these techniques and directly constraining the turnover of small bio-
molecules in situ using their hydrogen isotope composition, one that
could be applied to diverse environments.
Methods
Organic acid extraction
Organic acids were extracted following the procedure developed by
Mueller et al.
22
with minor changes to account for the high con-
centrations of chloride in the fracture waters. Brie
fl
y, samples of
fracture
fl
uid were titrated to pH >6 with NaOH if necessary. Sam-
ples were run through a Dionex Ag/H cartridge at 0.5 mL/min to
remove chloride after the cartridge had been washed with 300 mL
puri
fi
ed water (MilliQ) at 2 mL/min. The
fi
rst 0.5 mL of eluent from
the cartridge was discarded as it represented the dead volume. The
remaining sample was collected until almost all the resin was used,
carefully avoiding over-
fi
lling the cartridge, which would cause
chloride to leak through. The cartridge eluent was injected onto a
Dionex high performance ion chromatography instrument with an
AG-11HC column and a KOH gradient from 1 to 20 mM. The organic
acid fraction of the chromatogram was collected into vials using
manual fraction collection. This step was repeated for samples with
lower acetate concentration and collected into the same vial. The
collected acids were titrated to pH >6 with degassed, anoxic NaOH
and then dried down under nitrogen. Samples were redissolved in
LC-MS grade methanol.
Stable isotope analysis
The majority of samples were analyzed on a heated electrospray
ionization (HESI) Orbitrap QExactive HF (Thermo Fisher, Bremen,
Germany) following the protocol of Mueller et al.
22
. This technique
quanti
fi
es the molecular-average
δ
13
C (VPDB) and methyl-speci
fi
c
δ
2
H
of acetate by comparison to an working standard of sodium acetate
(
δ
13
C=
−
19.2
‰
,
δ
2
H=
−
127
‰
). Certain samples were measured on an
electrospray ionization (ESI) Orbitrap Exploris 240, but the mass
spectrometry parameters were identical and the same standard was
used for all measurements. Multiple sample introduction methods into
the Orbitrap were used throughout the course of this study.
For direct infusion measurements, 500
μ
Lsyringe(Hamilton)was
fi
lled with sample or standard solution (in LC-MS grade methanol) and
attached to a syringe pump (Chemyx). Solution was infused into the
mass spectrometer at 5
μ
L/min. After a 7-minute acquisition, the syr-
inge and its tubing were washed with 2 mL of LC-MS grade methanol
and the next sample or standard was loaded into the syringe pump.
This was repeated to achieve bracketed, sample-standard comparisons
(AAAABBBBAAAA, A = standard replicates, B = sample replicates). This
method was used when memory effects between sample and standard
due to large differences in
δ
2
Hor
δ
13
Cwereaconcern.Thiswas
especially important for
2
H-enriched acetate samples from exchange
experiments.
For dual inlet measurements, two 500
μ
L syringes (Hamilton)
were
fi
lled, one with sample and the other with standard solution (in
LC-MS grade methanol) and attached to a syringe pump (Chemyx).
The solution was infused into the mass spectrometer at 5
μ
L/min.
Using a Rheodyne 6-port valve, sample and standard were alter-
nated while achieving continuous
fl
ow of both (after Hilkert et al.)
44
.
Each acquisition block was 12 minutes with 4-5 minute switch times
between blocks cut out of the data acquisition to avoid carryover
effects. This was repeated to achieve bracketed, sample-standard
comparisons (ABABABA, A = standard replicates, B = sample repli-
cates). This method was used for the majority of Kidd Creek and
Birchtree samples. Acetate standard was diluted to match sample
ion current.
For in-
fl
ow injection measurements, samples were infused into
the mass spectrometer using a Vanquish Horizons HPLC Split
Sampler Autosampler and a Vanquish Horizons Pump set to 5
μ
L/
min with degassed LC-MS grade methanol as an eluent. An injection
volume of 50
μ
L was used to insert this sample into the
fl
ow of
methanol which carried it to the Orbitrap for 14 min. At that time the
fl
ow rate was increased to 30
μ
L/min to clear residual sample from
the transfer lines. At 18.5 minutes, the
fl
ow rate was dropped again
to 5
μ
l/min and after 90 s, the next injection began. Data acquisition
included all 20 min of the run but only integrated between 2 and
12 min to calculate isotope ratios. This was repeated to achieve
bracketed, sample-standard comparisons (ABABABA, A = standard
Article
https://doi.org/10.1038/s41467-024-53438-4
Nature Communications
| (2024) 15:9130
5
replicates, B = sample replicates). Acetate standard was diluted to
match sample ion current.
In all of the above methods, the following ESI parameters were
used as default. Minor adjustments were made daily to tune the
instrument for spray stability. Polarity = negative, spray voltage =
3.0 kV, spray current <0.2
μ
A, Auxiliary gas = 1 (arbitrary units), sweep
gas = 1 (arbitrary units), sheath gas = 10 (arbitrary units), auxiliary gas
temperature = 100 °C, RF lens = 60%, capillary temperature = 320 °C.
The following Orbitrap parameters were used for all analyses. Auto-
mated gain control = 1e6, resolution = 60,000, microscans = 1, quad-
rupole range = 57
–
62 m/z, lock mass = off. Raw data off the Orbitrap
was extracted using the software IsoX (Thermo Fisher, Bremen, Ger-
many) and converted to isotope ratios using a Python script. This
script uses the Makarov equation outlined in Mueller et al.
22
to convert
from ion intensities to ion counts. It then culls scans that are >99th
percentile or <1st percentile in total ion current to avoid integrating
scans with ion source aberrations.
Exchange reactions
High-temperature acetate-water exchange experiments were con-
ducted using a customized Dickson-type
fl
exible reaction cell setup
(Parr Instruments) with no vapor phase present. Each
fl
exible gold
bag was
fi
lled with 90 mL of 1 mM sodium acetate in MilliQ water
(pH 6-7) that was sparged with nitrogen and pressurized to 30 MPa.
Two experiments were performed at 150 °C in 5%
2
H
2
O. One was run
for a week, sampling every 24 h, while the other was run for a month,
sampling every 3
–
5 days. Another month-long experiment with 5%
2
H
2
O was performed at 100 °C, sampling every 3
–
5 days. Acetate-
water exchange experiments were also performed at 60 °C in 60 mL
serum vials. Each vial was
fi
lled with 50 mL of 1 mM sodium acetate
in 5% 2H
2
O (pH 7) that had been sparged with nitrogen and sealed
with a butyl rubber stopper and crimped with an aluminum cap. At
each timepoint, 1 mL of sample was collected via needle and syringe.
The sample was immediately frozen and stored at
−
20 °C and the
solution was sparged with nitrogen again to remove any air intro-
duced during sampling. These experiments were done in triplicate.
All exchange experiments were performed at pH 6
–
7tomatch
environmental conditions.
Additional high temperature
fl
exible gold bag experiments were
performed to determine the equilibrium isotope effect at 200 °C
(30 MPa). Each reaction cell was
fi
lled with 90 mL of 1 mM sodium
acetate (pH 6
–
7) in either
−
50
‰
or +110
‰
δ
2
H water. Each condition
was measured in duplicate, resulting in four total experiments. Sam-
ples were taken every hour for the
fi
rst six hours to measure the extent
of isotopic exchange with time and then every ~6
–
12 h for the next
66 h. At each time point, 1.5 mL of the sample was collected and dis-
carded to remove the dead volume from the sampling apparatus and
then an additional 1.5 mL of sample was taken for acetate
δ
13
Cand
δ
2
H
analyses. Collected aliquots were immediately frozen and stored at
−
20 °C until they were analyzed.
The kinetic rate constants for H-isotope exchange were calculated
using the formulation from Sessions et al.
43
:
F
e
F
t
F
e
F
i
=
e
kt
ð
1
Þ
where
F
t
is the
2
H fractional abundance (i.e., mole fraction) at a given
timepoint,
F
i
is the initial fractional abundance and
F
e
is the fractional
abundance at equilibrium. The latter was calculated using the frac-
tional abundance of the water and the equilibrium isotope effect from
DFT models at the corresponding temperature. In experiments where
the isotope composition approaches or reaches equilibrium, data
points close to the equilibrium value were discarded from the calcu-
lation of rate constant due to the large propagated errors when the
natural logarithm of the value
F
e
–
F
t
was close to zero.
Isotope fractionation calculations
The apparent hydrogen isotope fractionation between acetate and
water (
ε
acetate/water
) was calculated as:
2
α
acetate
=
water
=
δ
2
H
Acetate
+ 1000
δ
2
H
Water
+1000
ð
2
Þ
2
ε
acetate
=
water
=
2
α
acetate
=
water
1
× 1000
ð
3
Þ
Thermodynamics and cell density calculations
The free energy (
Δ
G) available to microbial metabolisms was calcu-
lated by adjusting the standard free energy (
Δ
G°) for the activity of the
reactants and products found in Kidd Creek fracture
fl
uids following
the equation:
Δ
G
=
Δ
G°
+
RT
lnQ
ð
4
Þ
where R is the ideal gas constant (kJ/mol/K) and T is temperature (K),
set to 298 K at 500 bar pressure. Q is the reaction quotient de
fi
ned as:
Q
=
Y
a
v
i
a
i
ð
5
Þ
where a is the activity of a substrate de
fi
ned as the product of its
concentration (molar) and gamma value and v is the stoichiometric
coef
fi
cient which is negative for reactants. Gamma values for sulfate,
methane and bicarbonate were found on the Geochemists Workbench
with the thermo-hmw.dat database, which uses a Pitzer equation based
Harvie-Møller-Weare activity model owing to the high ionic strength of
the fracture
fl
uid (4.9 molal). Acetate is not part of this database, so it
was calculated with extended Debye Hueckel equation using the
thermo.dat database. The concentrations used in these calculations
were taken from data in Lin et al.
10
. Sulfate, bicarbonate, acetate and
methane concentrations were set to 620
μ
M, 57
μ
M, 1.3 mM and
2.1 mM, respectively. Methane concentration was calculated from
fl
uid
fl
ow rate, gas exsolution rate from the
fl
uid, and the concentration of
methane in the gas (from Lin et al.
10
). It was assumed that all methane
was dissolved fully in solution due to the high (500 bar) in situ pressure
of the fracture
fl
uids (after Sherwood-Lollar et al.
11
). Sul
fi
de was below
detection limits (<2
μ
M). Its concentration was set to 10 nM but
increasing its concentration to the detection limit did not change the
implications of the cell densities ( > 10 cells/mL at all maintenance
energies simulated).
Cell density (cells/L) is calculated by combining the acetate
turnover rate (M/s), the free energy of the reaction (J/mol), and the
maintenance energy of a cell (J/s/cell).
ρ
=
τ
AC
×
Δ
G
ME
ð
6
Þ
where
τ
AC
is the turnover time and
ρ
is the cell density.
Density functional theory calculations of EIE
Temperature-dependent
2
H/
1
H equilibrium fractionation between
acetate and water was estimated using density functional theory.
Liquid-phase acetate and water molecular models were optimized in
the GAUSSIAN(TM) program, revision D.01 and GAUSSIAN 16, revi-
sion B.01 using basis set 6-311 G(d,p)
45
,
46
and functional B3LYP under
Tight optimization criteria (maximum/RMS atomic displacement
0.00006/0.00004 Bohr, maximum/RMS force 0.000015/0.00001
Hartrees/Bohr or Hartrees/Radian), with an Ultra
fi
ne integration grid
mesh. The integral equation-formalism polarizable continuum model
was used to represent the solvation environment
47
,
48
. Following
Article
https://doi.org/10.1038/s41467-024-53438-4
Nature Communications
| (2024) 15:9130
6
optimization, frequency calculations were carried out for the
monoisotopic isotopologues and with a single
2
H/
1
H substitution to
determine the effect of
2
H/
1
H substitution on vibrational frequencies.
The Urey-Bigeleisen-Mayer equation was used to calculate the
temperature-dependent reduced partition function ratio of each
species under
2
H/
1
H substitution
49
. Corrected ratios were computed
using the temperature-dependent regression of Wang et al.
48
to
account for the effects of anharmonicity
50
. The equilibrium fractio-
nation factor was then computed as the ratio of the corrected ratios
at the desired temperature.
Temperature-dependent
2
H/
1
H equilibrium fractionation between
the Ca-acetate complex and water was estimated using an empirically
derived molecular geometry for the complex, which was then opti-
mized using the same level of theory and basis sets as in the DFT
calculations above
51
. More details regarding these calculations can be
found in the Supplemental Information (Tables S4-S6). The partition
function ratio of water was adjusted to account for the
‘
salt effect
’
of a
3MCaCl
2
brine, which was empirically determined to be 15
‰
at 25 C by
Horita et al.
52
. The beta factor for water (
β
water
) was multiplied by 1.015
to ascertain the beta factor of the brine (
β
brine
):
β
brine
=1
:
015 ×
β
water
ð
7
Þ
The beta factors for free acetate (
β
acetate
) and Ca-acetate complex
(
β
Ca-acetate
), calculated from the DFT simulations, were then used to
calculate the EIE between acetate and water (
α
acetate/water
), between
acetate and brine (
α
acetate/brine
) and between the Ca-acetate complex
and brine (
α
Ca-acetate/brine
). For example, the EIE of Ca-acetate complex
and brine is calculated as such:
2
α
complex
=
brine
=
β
complex
β
brine
ð
8
Þ
2
ε
complex
=
brine
=
2
α
complex
=
brine
1
1000
ð
9
Þ
Radiolytic yield calculations
To estimate the radiolytic yield (nM/J) of acetate production by alpha,
gamma and beta irradiation in Kidd Creek needed to support a given
rate of acetate production, modi
fi
ed calculations from Warr et al.
9
were used. The total acetate yield (Y
AC
) in nM/s is de
fi
ned as:
Y
AC
=
P
E
net
,
i
×
G
i
×
ρ
bulk
φ
ð
10
Þ
where i represents either alpha, gamma or beta radiation and E
net
is the
dose rate (Gy/s) and G is the radiolytic yield (
G
). The bulk rock density
(
ρ
bulk
) was set to 2.98 kg/dm
3
.
φ
is the porosity, typically ~1% at crys-
talline rocks sites like Kidd Creek
9
. Here, we assume that beta and
gamma radiation does not produce acetate, since it has not been
measured, such studies have not yet been done, so only
α
radiation is
considered. Consequently, this represents a conservative estimate of
radiolytic acetate production. Alpha radiolytic yields were taken from
Vandenborre et al.
15
. In experiments with 200
μ
M dissolved carbonate
in pure water, acetate accumulated to 8
μ
M within 1400 Gy of absor-
bed radiation and plateaued at this concentration up to 5600 Gy, due
to competing production and consumption reactions reaching a
steady state. This results in a range of 1.3 to 6.0 nM/J for alpha radiation
yields.
The dosage rate of alpha radiation is calculated as:
E
net
,
α
=
X
E
α
,
X
×
W
×
S
α
1+
W
×
S
α
ð
11
Þ
Where
E
α
is the dosage of alpha radiation emitted (Gy/s) and
X
represents the speci
fi
c elemental source of that radiation.
S
α
is the
stopping power of rock to alpha radiation set at 1.5 after Warr et al.
9
.
W
is the water-rock ratio set to 0.37% calculated following Warr et al.
9
,
using water and rock density of 1.11 g/cm
3
and 2.98 g/cm
3
,respectively,
and a porosity value of 1%
5
,
9
.
At 1% K, 1 ppm Th and 1 ppm U, these elements emit 0, 1.93 × 10
−
12
and 6.9 × 10
−
12
Gy/s of alpha radiation, respectively
9
.ToestimateE
α
for
each of these elements in Kidd Creek, they were linearly increased
based on the actual concentration in the deposit, which are 1.5 ppm,
6.7 ppm and 1.7% for U, Th and K, respectively
9
. Therefore, the E
α
for U,
Th and K is estimated at 0, 1.3 × 10
−
11
and 1.0 × 10
−
11
Gy/s in Kidd Creek.
Reporting summary
Further information on research design is available in the Nature
Portfolio Reporting Summary linked to this article.
Data availability
The data
fi
les generated during ESI-Orbitrap analysis of the Kidd Creek
and Birchtree fracture
fl
uids is provided in a public GitHub repository
(
https://doi.org/10.5281/zenodo.13798759
)
53
. Any additional data
beyond those found in the repository can be made available on
request.
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Acknowledgements
We would like to thank Nathan Dalleska (Caltech) for helpful discussions
about sample processing as well as An
dreas Hilkert and Dieter Juchelka
(Thermo Fisher, Bremen) and the Caltech Proteome Exploration Lab for
use of their Orbitrap facilities. Funding for this work came from an NSF
Gradaute Research Fellowship DGE-1745301 (to E.P.M.), a European
Association of Organic Geochemistry Research Award (to E.P.M.), the
NASA Astrobiology Institute grant # 80NSSC18M0094 (to J.M.E. and
A.L.S.), a CIFAR Earth 4D grant (to B.S.L. and V.O). This work was also
supported by the Deutsche Forschungsgemeinschaft through the
Cluster of Excellence
“
The Ocean Floor
–
Earth
’
s Uncharted Interface
“
(project 390741603) to V.H.
Article
https://doi.org/10.1038/s41467-024-53438-4
Nature Communications
| (2024) 15:9130
8
Author contributions
E.P.M. conceptualized and designed the study and performed data
analysis. E.P.M., J.P. and M.S. perfo
rmed sample chemical preparation
and Orbitrap analysis. E.P.M., C.H. and V.H. performed isotope exchange
reactions. J.B. and A.M. performed D
FT calculations. J.E., A.L.S, V.O,
B.S.L, K.H., O.W. and W.B. provided laboratory analytical facilities and
samplesaswellasimportantscienti
fi
c insights. All authors contributed
to data interpretation and manuscript writing.
Competing interests
The authors claim no competing interests.
Additional information
Supplementary information
The online version contains
supplementary material available at
https://doi.org/10.1038/s41467-024-53438-4
.
Correspondence
and requests for materials should be addressed to
Elliott P. Mueller.
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Nature Communications
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