Radiolytically reworked Archean organic matter in a habitable deep ancient high-temperature brine

Investigations of abiotic and biotic contributions to dissolved organic carbon (DOC) are required to constrain microbial habitability in continental subsurface fluids. Here we investigate a large (101–283 mg C/L) DOC pool in an ancient (>1Ga), high temperature (45–55 °C), low biomass (102−104 cells/mL), and deep (3.2 km) brine from an uranium-enriched South African gold mine. Excitation-emission matrices (EEMs), negative electrospray ionization (–ESI) 21 tesla Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS), and amino acid analyses suggest the brine DOC is primarily radiolytically oxidized kerogen-rich shales or reefs, methane and ethane, with trace amounts of C3–C6 hydrocarbons and organic sulfides. δ2H and δ13C of C1–C3 hydrocarbons are consistent with abiotic origins. These findings suggest water-rock processes control redox and C cycling, helping support a meagre, slow biosphere over geologic time. A radiolytic-driven, habitable brine may signal similar settings are good targets in the search for life beyond Earth.

Table S2.Light element abundance and composition for the Moab Khotsong system, as well as their associated mass stopping powers and neutron yields for U and Th from Andrews et al. 3

14 C Production in Subsurface Fluids
Pathways of 14 C Contribution to the Moab Khotsong Brines Here we explore three major cases to explain 14 C introduction into noble-gas labeled 'ancient' fracture waters.Cases ( 1) and ( 2) are explored with equations of Heard et al. 4 for Wits Basin fracture waters.Case 3 presents a more in depth look at radiolytic contribution based on the texts of Andrews et al., 3 and Andrews et al., 5 .
Case 1: Contamination from air introduction during sampling: Estimate of the relative volumetric rate of air addition to the system: R = gas constant (8.2 ×10 -2 (L • atm)/K/mol) = temperature in K (in our case for 95-level, 54°C or 327K) 4.0 • 10 -. is the partial pressure of CO2 in air (in atm) Calculate the moles per liter addition of modern CO2 to sample groundwater: 0 =    = 0.112 L/min for 95-level *Fair value used here was 0.00060 L/min (upper contamination estimate) The amount of added CO2 that will dissolve into the sample water and be recorded as DIC: 0 = density of fluid in g/cm 3 = 1.04 g/cm 3 for the 95-level brine  +, $ % =Henry's constant for CO2 in molar Using estimated ages from measured ∆ 14 C: 95-level: 9,530 = -8033ln( % ) where  % = 30% contribution from modern fluid with modern 14 C 101-level: 8,250 =-8033ln( % ) where  % = 36% contribution from modern fluid with modern 14 C 1200-level: 6,410 = -8033ln( % ) where  % = (up to) 45% contribution from modern fluid with modern 14 C If this contribution from young water scales to contribution on noble-gas estimated residence times, this would result in an underestimate of the 95-level closed-system residence time by 30Ma.This can be accounted for in the error on noble gas derived residence times for this fluid system (±0.14 -1.87 Ga; Warr et al. 6 ).
Where  ' = Mass stopping power (6.8 MeV),  ' = the fractional abundance for light element i (Table S2), and  ' @ and  ' 7A are the neutron yields per µg/g of U and Th in equilibrium with their daughters.
Where K =  % • 220,000 [ % is the weighted mean absorption cross section, estimated at 0.0066 cm 2 g -1 ].  B multiplied by the velocity of a neutron (220,000 cm s -1 ) results in neutron flux () cm -2 s -1 for the system.In the case of Moab, this value was 9.53 • 10 -4 cm -2 s -1 .Decay of 14 N and 17 O were considered as the primary 14 C producing reactions in the Moab brine: 14 N + n => 14 C + p (reaction cross section 1.86 • 10 -24 cm 2 from Andrews et al. 3 ) 17 O + n => 14 C + a (reaction cross section 2.45 • 10 -25 cm 2 from Andrews et al. 3 ) Where  = cross section for the reaction considered, N = amount of parent species in atoms / cm 3 ,  ( = decay constant of 14 C (3.80 • 10 12 s), and t = time in s.Using estimates of 3.47 • 10 18 ( 14 N atoms / cm 3 ) and 2.67 • 10 19 ( 17 O atoms / cm 3 ) and, the total atomic 14 C production equaled 3.26 • 10 3 atoms cm -3 .Over the lifetime of the 1.2 Ga brine (assuming a closed-system), this production may account up to 7.5% modern 14 C.This value could be larger if the system receives more radionuclide contribution from the reef than estimated here.
Conclusions: There is likely significant in situ radiolytic contribution to 14 C in the Moab brines, explaining most of the age discrepancy between 14 C ages and noble gas estimated residence times for this system.This is due to contribution from the radiogenic rich gold reef in this system, and is supported by high noble gas radiogenic excesses, including the highest ever 86 Kr radiogenic excess found in subsurface fracture fluids (Warr et al. 6 ).

Fig. S1 .
Fig. S1.UV-Vis absorptivity of Moab Khotsong fluids.(a) Absorptivity vs. wavelength (200-700 nm) for Moab Khotsong fracture fluids and Vaal Reef sample.(b) Closer view of absorptivity vs. wavelength (200-380 nm) for Moab Khotsong brines and Vaal Reef Sample.UV-Vis spectra of both 95 and 101-level brines showed decreasing absorptivity with increasing wavelength, with no measurable absorptivity at wavelengths >250nm.Absorptivity values covered a range up to 0.03 L/(mg C • cm) between 200 and 240 nm, highlighting the presence of different organic species present in the brine DOC.

Fig. S2 .Fig. S3 .
Fig. S2.Excitation-Emission Matrices (EEMs) for 95-level brine sampled in 2018, 2019, and 2020.Spectra (a) with fluorescein, and (b) without fluorescein signature included.Scales represent fluorescence intensity.Contour lines for (a) are 0, 1, 10 for all samples.Contour lines for (b) are 0, 0.001, 0.01 for 2018 and 0, 0.05, 0.5 for 2019 and 2020 samples.The 95-level brine displayed a dominating fluorescence signature from the fluorescein dye throughout all  $% ranging from ~ 500 to 600 nm.Comparing 95-level DOC samples over time, there is a clear reduction in the fluorescein signal from 2018 to 2019, with a continued decrease into 2020.This provides additional evidence of borehole flushing and decreasing potential contamination from drilling fluid over the three-year sampling period for this sample.

Fig
Fig. S4.Percent relative abundance (%R.A.) of organic species.Includes all heteroatom classes identified through negative ion ESI 21 tesla FT-ICR MS for 95 and 101-level brines compared to the 1200-level fluid.FT-ICR MS spectra for samples shown are included in Supplementary Data 1.

Fig. S5 .
Fig. S5.Percent relative abundance (%R.A.) of organic species in major classes Ox, N1Ox, and SOx.Classifications obtained through negative ion ESI 21 tesla FT-ICR MS for 95 and 101-level brines compared to drilling additives and an MQ H2O Blank.If normalized for total sample DOC concentration, %R.A. peaks for MQ H2O (~1 mg C/L total DOC) would not be visible next to brine peaks.FT-ICR MS spectra for samples shown are included in Supplementary Data 1.

[ 3 . 4 •Case 2 :
10 -2 mol / (L • atm)]  +, $ 2'* =7.96 • 10 -8 mol/L or 7.96 * 10 -5 mmol/Là doesn't compare to concentrations of 0.266 or 1.07 mmol/L DIC for the 95 or 101levels Conclusion: Air contamination during sample is likely only a minor contributor to the 14 C content of the brines.*See note for DIC ages at the bottom of this section.*TheDIC ages of the Moab Khotsong brines were based on 14 C measurements taken on several year-old fluid samples collected in glass NOSAM bottles with greased stoppered caps.Over this time the grease likely dried out (based on analysis of other old Witwatersrand fluid samples stored in the same way), leading to younger 14 C based ages not captured in calculations of air contamination (Case 1), as those are dependent on gas and water flow rates at site.Contamination from mixing with a modern, surface-derived fluid: Consider an end-member mixing scenario where a young water component contains essentially modern 14 C and the DIC concentrations in both components are assumed to be equal and dominated by atmospheric recharge.

Relative abundance of organic species from negative ion ESI 21 tesla FT-ICR MS in 95 and 101-level brines vs. Vaal Reef.
Terms on the X-axis consist of the DBE number followed by molecular formula assigned to a given peak.The large peak at C17H26O4 for the Vaal Reef in (a) is a noise artifact and not an accurate representation of the sample composition.FT-ICR MS spectra for samples shown are included in Supplementary Data 1.