Pathways for abiotic organic synthesis at submarine hydrothermal fields

Significance

Arguments for an abiotic origin of organic compounds in deep-sea hot springs are compelling because of their potential role in the origin of life and sustaining microbial communities. Theory predicts that warm H₂-rich fluids circulating through serpentinizing systems create a favorable thermodynamic drive for inorganic carbon reduction to organic compounds. We show that abiotic synthesis proceeds by two spatially and temporally distinct mechanisms. Abundant dissolved CH₄ and higher hydrocarbons are likely formed in H₂-rich fluid inclusions over geologic timescales. Conversely, formate production by ΣCO₂ reduction occurs rapidly during subsurface mixing, which may support anaerobic methanogenesis. We confirm models for abiotic metastable organic compound formation and argue that alkanes in all ultramafic-influenced vents may form independently of actively circulating serpentinizing fluids.

Abstract

Arguments for an abiotic origin of low-molecular weight organic compounds in deep-sea hot springs are compelling owing to implications for the sustenance of deep biosphere microbial communities and their potential role in the origin of life. Theory predicts that warm H₂-rich fluids, like those emanating from serpentinizing hydrothermal systems, create a favorable thermodynamic drive for the abiotic generation of organic compounds from inorganic precursors. Here, we constrain two distinct reaction pathways for abiotic organic synthesis in the natural environment at the Von Damm hydrothermal field and delineate spatially where inorganic carbon is converted into bioavailable reduced carbon. We reveal that carbon transformation reactions in a single system can progress over hours, days, and up to thousands of years. Previous studies have suggested that CH₄ and higher hydrocarbons in ultramafic hydrothermal systems were dependent on H₂ generation during active serpentinization. Rather, our results indicate that CH₄ found in vent fluids is formed in H₂-rich fluid inclusions, and higher n-alkanes may likely be derived from the same source. This finding implies that, in contrast with current paradigms, these compounds may form independently of actively circulating serpentinizing fluids in ultramafic-influenced systems. Conversely, widespread production of formate by ΣCO₂ reduction at Von Damm occurs rapidly during shallow subsurface mixing of the same fluids, which may support anaerobic methanogenesis. Our finding of abiogenic formate in deep-sea hot springs has significant implications for microbial life strategies in the present-day deep biosphere as well as early life on Earth and beyond.

Seawater-derived hydrothermal fluids venting at oceanic spreading centers are a net source for dissolved carbon to the deep sea, with vent fluid carbon contents directly tied to the sustenance of the subseafloor biosphere (1). Highly reducing fluids rich in dissolved H₂, such as those discharging from serpentinizing hydrothermal systems, are of particular interest because of the potential for abiotic reduction of dissolved inorganic carbon $\left({\displaystyle \Sigma {\text{CO}}_2}={\text{CO}}_2+{\text{HCO}}_3^{-}+{\text{CO}}_3^{2-}\right)$ to organic compounds (2–6) and their potential role as precursor compounds for prebiotic chemistry associated with the origin of life (7). Although there is increasing evidence that supports an abiotic origin for CH₄ and other low-molecular weight organic compounds in ultramafic-hosted hydrothermal systems (8–10), the physical conditions, reaction pathways, and timescales that control abiotic organic synthesis at oceanic spreading centers remain elusive. Working models for the formation of abiotic CH₄ and other hydrocarbons observed in vent fluids involve reduction of ΣCO₂ and/or CO through Fischer–Tropsch-type processes during active circulation of seawater-derived hydrothermal fluids that are highly enriched in dissolved H₂ because of serpentinization of host rocks; however, this mechanism has not been conclusively shown in natural systems. Others have suggested that leaching of CH₄ and low-molecular weight hydrocarbons from magmatic fluid inclusions hosted in plutonic rocks may contribute at some level to the inventory of organic compounds observed in axial hot-spring fluids (1, 11, 12). The relative influence of these processes has important implications for the total flux and real-time concentrations of aqueous organic compounds delivered to the oceans by ridge-crest hydrothermal activity. Here, we use multiple lines of evidence to preclude abiotic reduction of ΣCO₂ to CH₄ during active fluid circulation but show that it is reduced to the metastable intermediate species formate instead.

Results and Discussion

Located at 2,350-m depth on the Mid-Cayman Rise (13, 14), hydrothermal fluids emanate from the Von Damm vent field at temperatures as high as 226 °C (Fig. S1). Ultramafic, gabbroic, and basaltic rocks are associated with the Mount Dent oceanic core complex that hosts this site (15–17). The highest temperature fluids venting at East Summit are characterized by high-dissolved H₂ (18.2 mmol/L), CH₄ (2.81 mmol/L), elevated C₂₊ hydrocarbons, low-dissolved metals, near-neutral pH (5.6), and near-zero concentrations of dissolved Mg (Fig. 1A, Table 1, and Fig. S2 A–C). Relative to seawater, dissolved Cl and ΣCO₂ abundances in the near-endmember East Summit fluids are slightly enriched, with concentrations of 651 and 2.80 mmol/kg, respectively (SI Text, section 1). Lower temperature fluids that contain substantial concentrations of Mg are also observed at the summit and around the flanks of the Von Damm mound. Aqueous concentrations of Cl, CH₄, ethane (C₂H₆), and propane (C₃H₈) in these fluids define single conservative mixing lines when each species is plotted against dissolved Mg, suggesting that these fluids have formed by subsurface mixing of cold Mg-rich ambient seawater with the same near-zero Mg fluids (18) sampled at the East Summit (Fig. 1A and Fig. S2 B–D).

Fig. S1.

Bathymetry of the Von Damm hydrothermal field located on the western flank of the Mid-Cayman Rise, with locations of fluid sampling indicated with circles.

Fig. 1.

Plots of measured Mg vs. (A) CH₄, (B) ΣCO₂, (C) ΣHCOOH, and (D) ΣCO₂ + ΣHCOOH concentrations for Von Damm vent fluids. Mg content is used as an indicator for seawater mixing; solid lines denote conservative dilution of the near-endmember composition (blue circles) with seawater (yellow stars), whereas dashed lines indicate species concentrations that result from nonconservative mixing in elevated Mg fluids (green symbols). Select δ¹³C_CO2 values are plotted in B near corresponding samples. Uncertainties (2σ) not shown are smaller than symbols.

Table 1.

Measured and calculated abundance and stable isotope data for Von Damm vent fluids

Vent	Sample	T (°C)	Mg (mm)	pH*	Cl (mm)	H2 (mM)	ΣHCOOH (μm)	ΣCO2 (mm)	CH4 (mM)	C2H6 (nm)	C3H8 (nm)	δ13CCO2 (‰)	δ13CCH4 (‰)	δ13CC2H6 (‰)	δ13CC3H8 (‰)
East Summit	Endmember	—	0	5.56	651	18.2	88.2	2.80	2.81	639	56	NA	NA	NA	NA
East Summit	J2-612-IGT2	226	2.93	5.65	649	16.2	82.0	2.79	2.62	603	52	0.8	−15.6	−12.9	−9.8
East Summit	J2-616-IGT8	226	2.43	5.56	641	18.3	85.6	2.75	2.72	—	—	0.9	−15.3	−12.3	—
White Castle	J2-616-IGT1	151	13.5	5.77	622	13.1	352†	2.51	2.08	485	41	1.5	−15.6	—	—
Ginger Castle	J2-617-IGT4	125	18.0	6.06	604	11.3	337†	2.35	1.88	—	—	2.2	−15.8	−13.2	−10.8
Ravelin 1	J2-617-IGT6	145	15.0	5.83	614	13.4	147†	2.52	2.02	—	—	1.9	−15.6	—	—
Ravelin 1	J2-617-IGT2	131	16.8	5.93	616	13.1	132	2.40	1.96	431	38	1.4	−15.1	—	—
Arrow Loop 1	J2-616-IGT6	134	18.5	5.86	616	10.8	274†	2.27	1.74	417	36	1.9	−15.7	−12.5	—
West Summit	J2-621-IGT1	123	24.0	6.00	605	9.94	428	2.08	1.64	359	30	3.3	−15.6	−12.6	—
West Summit	J2-621-IGT4	123	23.2	6.01	597	9.94	428†	2.07	1.67	335	29	3.6	−15.1	—	—
Ravelin 2	J2-621-IGT2	116	13.4	5.88	620	13.6	—	1.98	2.10	475	40	3.8	−15.1	−12.9	−9.7
Ravelin 2	J2-621-IGT8	115	22.0	6.12	600	10.9	474†	1.88	1.73	365	34	3.3	−15.4	−12.7	—
Old Man Tree	J2-612-IGT6	115	14.4	5.81	620	10.5	663	1.80	1.97	—	—	2.6	−15.2	—	—
Old Man Tree	J2-612-IGT8	114	14.0	5.89	621	10.2	669†	2.03	1.92	455	40	2.9	−15.0	−12.6	−11.6
Shrimp Hole	J2-617-IGT1	21	46.1	7.73	549	0.01	BD	2.01	0.29	51.8	4.6	1.1	−15.1	—	—
Bottom SW		∼5	52.4	∼8	545	0	∼1	2.25	0	0	0	1.1	NA	NA	NA

Analytical uncertainties (2σ) are ±2 °C for T; ±3% for Mg and Cl; ±5% for H₂, ΣHCOOH, ΣCO₂, CH₄, C₂H₆, and C₃H₈; ±0.05 units for pH; ±0.3‰ for δ¹³C_CO2; ±0.8‰ for δ¹³C_CH4; ±0.4‰ for δ¹³C_C2H6; and ±0.7‰ for δ¹³C_C3H8. Values that were not determined are indicated by —. BD, below detection (1.0 μm for ΣHCOOH); IGT, isobaric gas tight; mm, mmol/kg; mM, mmol/L; μm, μmol/kg; NA, not applicable; nm, nmol/kg; SW, seawater; T, temperature.

^*Shipboard pH is reported (25 °C and 1 atm).

^†Sample used to calculate measured affinities in Fig. 2.

Fig. S2.

Plot of measured Mg vs. measured (A) H₂, (B) C₂H₆, (C) C₃H₈, and (D) Cl. Conservative behavior in Cl, C₂H₆, and C₃H₈ as well as CH₄ in mixed fluids (green symbols; in the text) during mixing between near-endmember fluid (blue circles) and seawater (yellow stars) suggests that the Von Damm vent field is fed by a single-source fluid originating in the high-temperature reaction zone below the seafloor. Nonconservative H₂ behavior occurs at two mixed fluid vents: Old Man Tree (115 °C) and Shrimp Hole (21 °C). Uncertainties (2σ) not shown are smaller than symbols.

Elevated concentrations of dissolved H₂, CH₄, and low-molecular weight hydrocarbons are remarkably similar to abundances in other ultramafic-influenced hydrothermal systems (8–10, 19), consistent with a strong influence of serpentinization reactions in subseafloor reaction zones on the composition of Von Damm vent fluids. The carbon isotopic composition of dissolved CH₄ is uniform across the Von Damm vent field, with a δ¹³C value of −15.4‰ (Table 1). This value is significantly heavier than those typically associated with thermogenic CH₄ generation (−25‰ to −50‰) or microbial production of CH₄ from ΣCO₂ (−30‰ to −70‰) (20, 21), providing compelling evidence for an abiotic origin for Von Damm CH₄. An abiotic origin for CH₄ has been invoked for other ultramafic-influenced systems at Rainbow, Logatchev, and Lost City hydrothermal fields, where δ¹³C values for CH₄ range from −9‰ to −16‰ (8–10, 19).

The abundance and isotopic composition of aqueous carbon species in the Von Damm endmember fluids place important constraints on deep-seated processes responsible for the production of CH₄. Maximum fluid temperatures at Von Damm are at least 150 °C cooler than the predicted two-phase boundary for seawater at in situ seafloor pressures (22), suggesting that the minor Cl enrichment is not the result of subsurface phase separation (Fig. S3). Instead, the 19% enrichment in Von Damm endmember fluid Cl content (Fig. S2D) likely reflects the removal of water from seawater-derived fluids during serpentinization hydration reactions at low fluid to rock mass ratio (23). Applying a 19% correction to local bottom seawater ΣCO₂ concentrations (2.25 ± 0.11 mmol/kg) yields a predicted fluid ΣCO₂ abundance of 2.69 mmol/kg that matches, within analytical error, the observed endmember ΣCO₂ (2.80 ± 0.14 mmol/kg). Because the corrected ΣCO₂ abundance of the endmember fluid is nearly identical to that of ambient bottom seawater, we infer that no significant amounts of ΣCO₂ are added to or removed from the fluids during deep convective circulation before mixing in near-seafloor upflow zones. This argument is further supported by the δ¹³C isotopic composition of the endmember ΣCO₂ (0.9‰ ± 0.3‰), which is identical, within error, to that of local bottom seawater (1.1‰ ± 0.3‰). This conservation of ΣCO₂ during circulation through the crust has profound implications for the origin of the CH₄ in Von Damm vent fluids. With the addition of 2.81 mmol/kg CH₄, the endmember fluids contain approximately double the total carbon content of ambient seawater. Because the ΣCO₂ in the endmember fluids cannot provide the source of this carbon, this finding implies that CH₄ formation from reduction of inorganic sources is not occurring during active fluid circulation at the Von Damm site. Because CH₄ is the dominant product expected during abiogenic n-alkane synthesis (11), we infer that C₂₊ hydrocarbon formation is also not occurring during active fluid circulation.

Fig. S3.

Plot of maximum sampled temperature and seafloor pressure conditions at the Von Damm vent field (blue circle). The curve represents the two-phase boundary of seawater (22). To attain measured temperatures, Von Damm fluids would have cooled by at least 150 °C after phase separation.

Radiocarbon analysis provides additional confirmation that Von Damm CH₄ is not derived from fluid ΣCO₂ contents. The four Von Damm CH₄ samples measured, including the East Summit fluid, all reveal ¹⁴C contents near the detectable limit [fraction modern (F_m) = 0.0025] (Table S1). In contrast, corresponding ΣCO₂ samples contain detectable modern ¹⁴C contents (F_m = 0.0236–0.0373) that would be transferred to CH₄ if it were generated by ΣCO₂ reduction occurring during fluid circulation (Table S1). Thus, the model postulated for the formation of abundant CH₄ at the Lost City vent field involving the leaching of radiocarbon-dead ΣCO₂ from fluid inclusions hosted in plutonic rocks and its subsequent reduction to CH₄ during hydrothermal fluid circulation (8) cannot account for the occurrence of CH₄ at Von Damm.

Table S1.

Radiocarbon data for the Von Damm vent fluids

Vent	Sample	Measured CH4 (Fm)	Measured ΣCO2 (Fm)	Corrected ΣCO2 (Fm)	Accession nos.
East Summit	J2-612-IGT2	0.0064 ± 0.0013	0.0660 ± 0.0015	0.0251 ± 0.0046	OS-104460 (CH4), OS-105946 (ΣCO2)
East Summit	J2-616-IGT8	0.0056 ± 0.0013	0.0712 ± 0.0050	0.0373 ± 0.0069	OS-104461 (CH4), OS-104700 (ΣCO2)
Ravelin 2	J2-621-IGT2	0.0051 ± 0.0013	—	—	OS-104462 (CH4)
Ravelin 2	J2-621-IGT8	0.0074 ± 0.0023	0.3573 ± 0.0029	0.0236 ± 0.0395	OS-104706 (CH4), OS-104344 (ΣCO2)

IGT, isobaric gas tight.

In contrast, we suggest that CH₄ and C₂₊ hydrocarbons in the Von Damm vent fluids are derived from leaching of carbon-rich fluid inclusions at depth. We postulate that the abundant CH₄, C₂H₆, and C₃H₈ in Von Damm vent fluids were formed when magmatic volatiles trapped in plutonic rocks reequilibrated during cooling to temperatures <400 °C, generating hydrocarbon-rich and ΣCO₂-poor fluid–vapor inclusions as described for CH₄-rich Southwest Indian Ridge gabbros (12, 24). We propose that, at Von Damm, these hydrocarbons are subsequently liberated during hydrothermal alteration of the Mount Dent oceanic core complex host rocks (15–17). CH₄ observed in Southwest Indian Ridge plutonic rock fluid inclusions is characterized by δ¹³C values of −10‰ to −30‰ (1, 25), a range that matches the values for not just the Von Damm field (−15.4‰) but also, all previously studied ultramafic-influenced submarine hydrothermal fields (8–10). This observation, again, supports our arguments that the processes that we reveal here may be directly relevant to all such systems.

Another line of evidence supporting a magmatic volatile-rich fluid inclusion input is the isotopic composition of He in the Von Damm vent fluids, which indicates R/R_a values of 8.0–8.2 that are consistent with a mantle source (26) (Table S2). Measured CH₄/³He ratios (∼2.4 × 10⁸) that are just below the average value of ΣCO₂/³He measured in mantle rocks (1 × 10⁹) (26) also support a mantle-derived (fluid inclusion) source for the hydrocarbons. This CH₄/³He ratio suggests a conversion of ∼24% of mantle-derived carbon to CH₄. Formation of graphite, which can precipitate on cooling of plutonic fluid inclusions (12), may account for the remainder of the carbon.

Table S2.

He isotope data for the Von Damm vent fluids

Vent	Sample	Measured 3He/4He (R/Ra)	Measured 4He (mL STP/g)	Measured 4He (μmol/kg)	Calculated 3He* (μmol/kg)	Measured CH4 (μmol/kg)	Measured CH4/3He	Predicted C† (μmol/kg)	Measured CH4/predicted C (% converted)
East Summit	J2-616-IGT8	8.209 ± 0.131	2.477E−05	1.01	1.147E−05	2,715	2.367E+08	11,473	0.24
Ravelin 1	J2-617-IGT6	8.265 ± 0.133	1.871E−05	0.76	8.721E−06	2,020	2.316E+08	8,721	0.23
Ginger Castle	J2-617-IGT4	7.956 ± 0.130	1.785E−05	0.73	8.012E−06	1,875	2.340E+08	8,012	0.23

IGT, isobaric gas tight; STP, standard temperature and pressure.

^*Calculated from ⁴He and R/R_a.

^†Predicted from ³He and the standard magmatic CO₂/³He value of 1 × 10⁹.

Thermodynamic models for the abiotic synthesis of aqueous organic compounds under hydrothermal conditions have postulated that kinetic barriers to the formation of CH₄ preclude stable equilibrium in the C-H-O chemical system, thereby creating a thermodynamic drive for the formation of metastable organic species in submarine hot springs (3, 4). The abundance of aqueous carbon species more oxidized than CH₄ in Von Damm vent fluids supports such a model. For example, in contrast to dissolved CH₄ concentrations, concentrations of ΣCO₂ in lower temperature mixed fluids at Von Damm are depleted by as much as 25% relative to a conservative mixing assumption. These depletions are accompanied by ¹³C enrichment of the residual ΣCO₂ compared with the endmember vent fluids and seawater (Fig. 1B and Table 1) and significantly enriched formate species (ΣHCOOH = HCOOH + HCOO⁻) abundances of 73–605 μmol/kg relative to conservative mixing (Fig. 1C). This result suggests that abiotic ΣHCOOH formation represents a sink for vent fluid ΣCO₂ in subsurface mixing zones. Consistent with this interpretation, the amount of carbon present as ΣHCOOH and ΣCO₂ in the endmember fluid at East Summit remains constant during mixing in the cooler fluids (Fig. 1D). Despite a strong thermodynamic drive, CH₄ production from ΣCO₂ does not occur because of well-established kinetic limitations, permitting the formation of metastable intermediate ΣHCOOH species. Reduction of ΣCO₂ by H₂ in mixed fluids (CO₂ + H₂ = HCOOH) is consistent with thermodynamic predictions in the absence of CH₄ production and laboratory experiments that have shown rapid reaction kinetics over hour to day timescales (2, 6) and isotopic enrichment of the residual ΣCO₂.

Thermodynamic Evaluation of ΣHCOOH Abundances.

Fluid compositions are consistent with metastable thermodynamic equilibrium between ΣCO₂, ΣHCOOH, and H₂ in Von Damm mixed fluids, providing additional support for an abiotic origin. Formation of ΣHCOOH on mixing represents a move to a near-equilibrium condition as indicated by decreasing chemical affinities that reach values below 5 kJ/mol for most of the sampled fluids (Fig. 2 and SI Text, section 2). Thus, unlike CH₄, the absence of kinetic barriers allows for abiotic synthesis of ΣHCOOH in subsurface mixing zones during active circulation of submarine hydrothermal fluids.

Fig. 2.

Chemical affinity for the production of HCOO⁻ from ΣCO₂ and H₂ in Von Damm mixed fluids. White symbols indicate a thermodynamic drive for reaction (positive affinity) as written based on conservative dilution of the near-endmember ΣHCOOH composition (blue circle). Green symbols denote affinity calculated with actual mixed fluid ΣHCOOH contents. Thermodynamic equilibrium is defined as affinity = 0 ± 5 kJ/mol (light blue shading). Symbol shapes correspond to those in Fig. 1.

Because of kinetic inhibition of CH₄ formation, there is a thermodynamic drive for the abiotic production of other metastable low-molecular weight organic species in addition to ΣHCOOH. Although methanol (CH₃OH) production from ΣCO₂ and H₂ is thermodynamically favorable at Von Damm, the predicted metastable equilibrium CH₃OH abundances in mixed fluids would be greater than predicted ΣCO₂ abundances, exceeding 2 mmol/kg. Our observation that the amounts of carbon present as ΣHCOOH and ΣCO₂ remain constant during mixing (Fig. 1D), therefore, precludes equilibrium CH₃OH formation. Similarly, abiotic production of other carboxylic acids (e.g., acetic, propanoic, and butanoic) is likely kinetically inhibited, because these species are below detection (< 1 μmol/kg). The organosulfur compound methanethiol (CH₃SH) is present in Von Damm fluids at abundances that do not reflect metastable equilibrium with ΣCO₂ and has been attributed to thermal alteration of microbial biomass or other sources of preexisting organic matter (27). The presence of other potential metastable species, such as amino acids, was not investigated; however, if they formed on mixing, it is likely that their concentrations are below the precision of ΣCO₂ analysis (∼100 μmol/kg).

Implications.

Vent microorganisms inhabit environments dominated by mixed hydrothermal fluids, where ΣHCOOH can be used as an energy or fixed carbon source by methanogenesis. With abundances approaching those of ΣCO₂, ΣHCOOH-based methanogenesis could be a viable metabolic strategy at Von Damm, and abiotic ΣHCOOH may represent an important substrate for microorganisms in high-H₂ hydrothermal fluids, which was hypothesized previously for Lost City (28, 29). Formate is the first intermediate species formed in the acetyl-CoA pathway (30), and its abiotic production can reduce the energetic demand for an organism, while also serving as the first step toward forming reduced carbon species that were central to primitive biochemical pathways on early Earth (31). Our demonstration of abiogenic production of CH₄ and likely, C₂₊ n-alkanes in deep-sea hydrothermal systems is also relevant to understanding metabolic options on early Earth environments as well as life strategies in modern systems.

Fluid circulation at Von Damm integrates abiotic organic species formed on long as well as short timescales. Hydrothermal fluids are rich in CH₄ leached from ancient magmatic volatile fluid inclusions hosted in plutonic rocks, where it is formed over geologic timescales, and ΣHCOOH, which is formed actively during shallow subsurface mixing over hours to days. These findings represent a fundamental advance in our understanding of processes leading to abiotic organic synthesis in modern and ancient systems on Earth as well as other planetary bodies (7, 31, 32). Furthermore, the demonstration of ongoing ΣHCOOH synthesis is important for microbial communities in the present-day oceanic crust, with exciting implications for microbial metabolisms and life strategies in any warm high-H₂ natural waters.

SI Text

1. Calculation of Endmember Compositions.

An endmember composition is calculated for the 226 °C East Summit fluid following the established practice in hydrothermal chemistry of regressing individual chemical species to zero Mg content based on studies that show near-quantitative Mg removal in high-temperature fluids (Table 1) (18). For some species (e.g., ΣCO₂ and ΣHCOOH), lower temperature (21–151 °C) mixed fluids exhibit nonconservative behavior that is the result of processes occurring during mixing between an endmember fluid and seawater in the subsurface before their expression at the seafloor. Because extrapolation to zero-Mg endmembers would not be meaningful for nonconservative species, their concentrations are considered in terms of measured abundances only.

2. Assessment of HCOOH Metastable Equilibrium Using Chemical Affinity.

The equilibrium state of the reaction

$${\text{CO}}_2+{\text{H}}_2={\text{HCOO}}^{-}+{\text{H}}^{\mathrm{+}}$$ [S1]

in Von Damm mixed fluids can be assessed by calculating the chemical affinity (A) defined by the relationship

$$A=-{\mathrm{\Delta }}_{r}G=-RT\ln \left(Q_r/K_{eq}\right),$$ [S2]

where R is the universal gas constant, T is measured fluid temperature (Kelvin), Q_r is the reaction quotient, and K_eq is the equilibrium constant at T and seafloor pressure (230 bar). In situ pH values ranging from 5.7 to 5.9 were calculated at seafloor pressure and sampled maximum temperature using thermodynamic data compiled in the SUPCRT92 database (41, 42) and the EQ3/6 software package (43, 44). Activity coefficients were calculated in EQ3/6 for HCOO⁻ and assumed to be unity for neutral dissolved species.

Materials and Methods

Vent fluid samples were collected using 150-mL titanium isobaric gas-tight samplers (33) deployed by the remotely operated vehicle Jason II aboard the R/V Atlantis (Cruise AT18-16) in January of 2012. Thermocouples were calibrated with a National Institute of Standards and Technology temperature calibrator, and the maximum measured temperature for each sample is reported (Table 1). Samples were extracted and processed within 24 h of sampler recovery. Immediately after withdrawing the fluid aliquot from the isobaric gas-tight sampler, pH (25 °C and 1 atm) was measured by potentiometry using an Ag/AgCl reference electrode. Aliquots were collected in Optima HCl-cleaned high-density polyethylene bottles for shore-based analysis of Mg, Cl, and total formate species (ΣHCOOH = HCOOH + HCOO⁻) in samples stored frozen. Shipboard measurement of dissolved H₂ and CH₄ was accomplished by molecular-sieve gas chromatography (GC) with thermal conductivity detection after a headspace extraction (3, 34). Aliquots for shore-based total dissolved inorganic carbon (ΣCO₂ = CO₃²⁻ + HCO₃⁻ + H₂CO₃) abundance and stable and radiocarbon isotope analysis of CH₄ and ΣCO₂ were transferred to evacuated 25-mL serum vials poisoned with Hg₂Cl and sealed with butyl rubber stoppers that were preboiled in NaOH to remove trace hydrocarbons (35). Dissolved He was extracted from fluid samples on board the ship using a portable vacuum line and transferred to evacuated aluminosilicate glass break-seal tubes for shore-based He isotope analysis. Fluid aliquots were transferred into sealed glass tubes fitted with Teflon and stainless steel valves for shore-based C₂H₆ and C₃H₈ analysis using a purge and trap device interfaced to molecular-sieve GC with flame ionization detection (34). Dissolved Cl and ΣHCOOH abundances were determined by ion chromatography (36, 37). Dissolved Mg concentrations were determined on a ThermoElectron Element2 inductively coupled plasma mass spectrometer (MS) (36, 38). Dissolved ΣCO₂ abundances were determined by headspace gas GC injection with thermal conductivity detection (34, 36). Stable carbon isotopes (δ¹³C_ΣCO2 and δ¹³C_CH4) were measured by isotope ratio monitoring MS using a Finnigan DeltaPlusXL Mass Spectrometer coupled to an Agilent 6890 GC (1,150 °C combustion temperature). Stable carbon isotope data are reported in standard δ-notation (δ¹³C) expressed as

$${\delta }^{13}\text{C}(‰)=\left[\frac{R_{samp}-R_{std}}{R_{std}}\right]\times \mathrm{1,000},$$ [1]

where R_samp and R_std are the isotope ratios (¹³C/¹²C) of the sample and the standard, respectively. Carbon stable isotopes are reported relative to the Vienna Pee Dee Belemnite Scale. Because of variable entrainment of ambient seawater that contains 2.25 mmol/kg CO₂ with a δ¹³C_ΣCO2 value of 1.1‰, reported sample δ¹³C_ΣCO2 values have been calculated from measured values using isotope mass balance (34). Analytical uncertainties (2σ) in abundance and isotopic analyses are listed in Table 1. Radiocarbon (¹⁴C_ΣCO2 and ¹⁴C_CH4) analysis was conducted at the Woods Hole Oceanographic Institution National Ocean Sciences Accelerator Mass Spectrometry Facility (Table S1). Results are expressed in terms of F_m, representing the deviation of the sample relative to the modern National Bureau of Standards Oxalic Acid I standard (NIST-SRM-4990; A.D. 1950) (39). Correction of ΣCO₂ radiocarbon measurements (Table S1) removes the effects of entrainment of ambient seawater through an isotopic mass balance approach that is analogous to the approach for δ¹³C_ΣCO2. [For example, this calculation uses vent fluid [Mg] as measured, East Summit fluid [ΣCO₂] as measured, Ravelin 2 fluid [ΣCO₂] assuming conservative endmember–seawater mixing (i.e., before ΣHCOOH formed), seawater [Mg] = 52.4 mmol/kg, seawater [ΣCO₂] = 2.25 mmol/kg, and seawater F_m = 0.9300 (∼580 y, estimated from 2,500-m depth; World Ocean Circulation Experiment Caribbean line A22, 1997).] Measured analytical uncertainties are listed in Table S1. Corrected ΣCO₂ uncertainties are conservative estimates calculated by error propagation of independent variables (e.g., also taking into account the effects of [Mg] and [ΣCO₂] analytical uncertainties). He abundance and isotope compositions were determined at the Isotope Geochemistry Facility at Woods Hole Oceanographic Institution (Table S2). Helium was cryogenically separated from the other noble gases (40), and analyzed as described in the work by German et al. (13). Uncertainties for ⁴He abundances are approximately ±5% because of splitting procedures (Table S2).