Abstract EANA2025-191

Phosphorus is a fundamental element for life, central to biomolecules such as DNA, RNA, ATP, and cellular membranes. However, the availability and mobility of phosphorus in subsurface environments, both on Earth and potentially on other planetary bodies, remain poorly understood and are likely limited and controlled by complex biogeochemical processes. The deep terrestrial biosphere, specifically that found in the Moab Khotsong mine (at ~3 km depth), is characterized by energy-limited, oligotrophic conditions, radiolytic hydrogen/oxygen production, microoxic and hypersaline environments, and elevated pressure and temperature. This unique setting serves as a valuable analog for assessing life’s potential and signatures of habitability in the subsurface environments of Mars, and other extraterrestrial bodies.

In this study, we investigate the distribution, diversity, and functional potential of phosphorus-cycling genes within bacterial communities inhabiting deep subsurface ecosystems spanning a geological profile from 1.2 to 3.2 km depth, where bioavailable phosphorus is undetectable. By integrating in situ enrichment, metagenomic sequencing, and the curated phosphorus-cycling gene database PCycDB, we identified key genes involved in phosphorus acquisition, solubilization, redox transformations, storage, and metabolism, including those associated with phosphonate biosynthesis and catabolism. These genes were detected in a deep subsurface biofilm growing on a channel receiving fracture water at 1.2 km depth, as well as in 13 isolates isolated from hypersaline brine at 3.2 km depth. Our results reveal that two distinct bacterial communities inhabit the fracture water and the hypersaline brine. Sulphate-reducing bacteria (e.g., Desulfomonile, MSBL7) and sulphide-oxidizing taxa (e.g., Thiothrix, Thiavirga) were predominant in the biofilm, whereas the isolates from the hypersaline brine belong to the genera Halomonas, Chromohalobacter, Bacillus subtilis, and Cytobacillus.

In both the biofilm and the isolates, genes associated with nucleotide reworking and turnover (e.g., purF, spoT, nrdA, nrdE)  along with those involved in the phosphorus starvation response (e.g., phoB, phoR), were among the most abundant. This pattern likely reflects the extreme phosphorus limitation in these environments, as neither inorganic nor organic phosphate was detected using ion chromatography or 31P nuclear magnetic resonance (NMR) spectroscopy. The alkaline conditions of the fracture and brine waters at both depths may facilitate the immobilization of phosphate as hydroxyapatite, thereby significantly reducing the availability of bioavailable phosphorus. Notably, genes (e.g., phoD) associated with organic phosphoester hydrolysis were identified at both depths. These genes encode enzymes that cleave organic phosphorus compounds, releasing inorganic phosphate (Pi) that can be assimilated by microorganisms. This finding aligns with the detection of genes involved in phosphonate biosynthesis (e.g., pepM) in the biofilm and phosphonate catabolism (e.g., pbfA, phnY) in the isolates and biofilm, indicating the presence of multiple microbial strategies for accessing alternative phosphorus sources under nutrient-limited conditions. The detection of phosphonate biosynthesis genes in the terrestrial deep subsurface is particularly significant, as it suggests a previously unrecognized biogeochemical pathway. Microbial production of phosphonates may serve as a biosignature for evaluating subsurface habitability on Earth and beyond. These findings highlight the metabolic versatility of deep biosphere microorganisms and offer insights into alternative phosphorus-utilization strategies that may be relevant to life in phosphorus-limited extraterrestrial environments.