Reduction spheroids preserve a uranium isotope record of the ancient deep continental biosphere

Life on Earth extends to several kilometres below the land surface and seafloor. This deep biosphere is second only to plants in its total biomass, is metabolically active and diverse, and is likely to have played critical roles over geological time in the evolution of microbial diversity, diagenetic processes and biogeochemical cycles. However, these roles are obscured by a paucity of fossil and geochemical evidence. Here we apply the recently developed uranium-isotope proxy for biological uranium reduction to reduction spheroids in continental rocks (red beds). Although these common palaeo-redox features have previously been suggested to reflect deep bacterial activity, unequivocal evidence for biogenicity has been lacking. Our analyses reveal that the uranium present in reduction spheroids is isotopically heavy, which is most parsimoniously explained as a signal of ancient bacterial uranium reduction, revealing a compelling record of Earth’s deep biosphere.


Supplementary Note 1: Uranium isotope systematics and enzymatic reduction
Uranium isotopes (U 238 /U 235 : δ 238 U) have been developed as a low-T paleo-redox proxy for the Earth's surface and upper crustal environments 1,2,3,4,5,6 . A thorough review of non-radiogenic U isotope fractionation has been recently published 7 but relevant material is summarized here.
The uranium isotope composition of crustal (and high-T) rocks shows a relatively narrow range of values averaging -0.29 ± 0.03‰ (ref. 8). In the modern Earth surface, biologically-mediated reduction of U in sediments under marine sub-oxic or euxinic bottom waters, or in pore fluids represents the biggest sink for uranium 2,5,9,10 .
U isotope fractionation is thought to be controlled by the nuclear volume effect, with the most significant fractionation occurring during U(VI) reduction to U(IV), concentrating the heavy 238 U isotope in the reduced product 1,2,5,10,11 . Although theoretical predictions verified by chromatographic isotope-separation experiments suggest that redox equilibration should enrich U(IV) in 238 U abiotically 12,13 , at low temperatures this effect has only been observed (so far) where reduction is biologically (enzymatically) mediated, probably for kinetic reasons 10 . Indeed, recent work 10 suggests that abiotic reduction by reduced mineral species and other natural reductants either does not fractionate or causes the opposite fractionation to biological reduction 1,10,14,15,16 . Based on this experimental evidence, ref. 10 suggested that U isotope fractionation in this way represented a proxy for biological reduction of uranium in natural low-temperature settings.
Isotopically heavy uranium is also found in magmatic titanite and zircon crystals as a consequence of high-temperature abiotic processes 17 , and in hydrothermally altered ocean basalts and associated calcium carbonate veins, where it might reflect either biotic or abiotic fractionation processes 18 . However, the factors controlling uranium isotope fractionation in these systems are quite unlike those in the reduction spheroids analysed here. By contrast, sandstone-hosted roll front uranium ore deposits are genetically similar to reduction spheroids; both systems represent sharp redox boundaries occurring at low temperatures in the continental subsurface. It has recently been shown 19 that the uranium in roll front deposits is isotopically heavy as a consequence of enzymatic uranium reduction, validating the proposal of ref. 10 and demonstrating the feasibility of the results obtained in the present study.

Supplementary Note 2: Additional geochemical data
Carbon isotope data were measured at the Scottish Universities Environmental Research Centre. The (solid hydrocarbon) cores from spheroids at Dingwall yielded δ 13 C PDB values of -43.0, -44.0 and -44.0. Organic-rich cores from Heysham yielded values of -29.5 and -31.1. These values have been affected by ionizing irradiation 20,21,22 and in any case do not reflect the biogenicity of the spheroids themselves because the organic material pre-dates spheroid formation.
GC-MS analysis of the spheroid cores from Dingwall (Supplementary Figure 1) revealed the presence of hopane and sterane biomarkers consistent with biogenic hydrocarbons 22 . However, such biomarkers do not demonstrate that the reduction spheroids themselves were produced by microbial metal reduction, since the organic matter pre-dates the existence of the spheroids.
Activity ratios ( 234 U/ 238 U) are given in Supplementary Table 1 and illustrated in Supplementary Fig. 2. Deviations from an activity ratio of 1 can be caused by incomplete digests (fractionating 234 U, but not 235 U or 238 U). Our digest method will not result in quantitative dissolution of all U bearing minerals (e.g., zircons). Alternatively, recent (<2Ma) interaction with oxidising or reducing fluids (e.g., groundwater) can also remove or add 234 U to sediments; the decay of this isotope causes alpha-recoil damage to the mineral lattice which allows 234 U to escape preferentially. Though many of our samples are within error of secular equilibrium, some cores of reduction spots are depleted in 234 U, and some halos are enriched. Overall, we observe no significant correlation between ( 234 U/ 238 U) and δ 238 U (r 2 =0.39). Non-equilibrium values are observed most commonly in samples from Budleigh Salterton, but matrix samples from Budleigh Salterton do not show much deviation from secular equilibrium. This suggests a relatively closed system in which the very high concentration of uranium present in the cores promoted localised lattice damage and the leaching, diffusion or ejection of 234 U from the cores to the halos, but not beyond the halos (cf. ref. 20); such a process need not require recent interaction with groundwater and could perhaps be ancient. In any case, the abundance of uranium would minimise the effect of any recent overprinting on the overall δ 238 U values, which we therefore consider most likely to record early diagenetic processes in the ancient deep biosphere.