Radiolytic support for oxidative metabolism in an ancient subsurface brine system

Abstract

Long-isolated subsurface brine environments (Ma-Ga residence times) may be habitable if they sustainably provide substrates, e.g. through water-rock reactions, that support microbial catabolic energy yields exceeding maintenance costs. The relative inaccessibility and low biomass of such systems has led to limited understanding of microbial taxonomic distribution, metabolism, and survival under abiotic stress exposure in these extreme environments. In this study, taxonomic and metabolic annotations of 95 single-cell amplified genomes were obtained for one low biomass (10³-10⁴ cells/ml), hypersaline (246 g/L), and radiolytically enriched brine obtained from 3.1 km depth in South Africa's Moab Khotsong mine. The majority of single-cell amplified genomes belonged to three halophilic families (Halomondaceae (58%), Microbacteriaceae (24%), and Idiomarinaceae (8%)) and did not overlap with any family-level identifications from service water or a less saline dolomite aquifer sampled in the same mine. Functional annotation revealed complete metabolic modules for aerobic heterotrophy (organic acids and xenobiotic oxidation), fermentation, denitrification, and thiosulfate oxidation, suggesting metabolic support in a microoxic environment. Single-cell amplified genomes also contained complete modules for degradation of complex organics, amino acid and nucleotide synthesis, and motility. This work highlights a long-isolated subsurface fluid system with microbial metabolism fueled by radiolytically generated substrates, including O₂, and suggests subsurface brines with high radionuclide concentrations as putatively habitable and redox-sustainable environments over long (ka-Ga) timescales.

Keywords: hypersaline brine; microbial diversity; radiolysis; single-cell amplified genomes; subsurface biosphere.

Figure 1

Images of cells via optical microscopy for 95-level (A), and via fluorescence microscopy combined with SYTO-9 staining for the (B) 101-level, (C) 1200-level, and (D) service water. Scale bar on the lower right of each image represents 5 μm. Magnification was 50x for (A) and 1000x for (B), (C), and (D). Cells in (A) are circular features entrapped in halite fluid inclusions and identified by arrows. Cells in (B), (C), and (D) appear as rod or spiral shaped objects. In (B), background fluorescence highlights the high level of DOC debris for this sample [28]. Cell counts for the 95-, 101-, 1200-levels and service water were 10² cells/ml ± 10, 10⁴ cells/ml ± 10², 10⁶ cells/ml ± 10^4.5, 10⁵ cells/ml ± 10^3.3, respectively (error in standard deviation). A schematic is included to the right showing the depth and major lithology of fluid sampling sites considered in this study.

Figure 2

Percent relative abundance (%) of GTDB-Tk-identified families in 101-level brine SAGs, 1200-level MAGs and service water MAGs. Shared taxa between the 1200-level and service water are shown with a dashed border, which could indicate infiltration of contaminating service water microorganisms not native to the 1200-level community. Family taxonomic names are distinguished by bolded phylum-level groups in the legend.

Figure 3

Total community nutrient cycling diagrams for 101-level brine SAGs as determined by METABOLIC-C for (A) carbon, (B) nitrogen, and (C) sulfur. Red arrows indicate the metabolic function is present in at least one SAG in the community (≥75% of gene annotations found for that function in a SAG). Symbols indicate genera associated with a particular pathway, based on individual nutrient cycling annotations for the top three most complete SAGs in each genus, including Agrococcus (right triangle), Atopococcus (three-quarter circle), Caenispirillum (circle), Chromohalobacter (diamond), Curtobacterium (trapezoid), Halomonas (square), Idiomarina (star), Microbacterium (x mark), Pseudomonas (equilateral triangle), and Shinella (plus sign).

Figure 4

Presence/absence of KEGG metabolic modules for 101-level SAGs. Black squares indicate a module is present for a particular SAG (>70% of KOFAM annotations are present for the identified KEGG metabolic module). Colored categories on the right indicate the broader KEGG functional category for each module. SAGs are positioned in the tree relative to module presence or absence.

Figure 5

Survivability (in years) for E. coli, B. subtilis, and D. radiodurans under accumulated radiolytic dosage. These represent illustrative examples of how specific organisms would respond if exposed to these doses. A high radionuclide concentration scenario (100 μg/g) and a low contribution scenario (1 μg/g) were considered along with three scenarios of radionuclide type (100% alpha radiation, 100% gamma radiation, and 50% alpha with 50% gamma radiation). Black horizontal lines represent the accumulated dosage at which a 10⁻⁶ survival fraction is reached for E. coli (1000 Gy) [56], B. subtilis (8400 Gy) [55], and D. radiodurans (15 000 Gy) [56].