Abstract EANA2026-73

On Earth, recently formed lava tubes are pristine, oligotrophic environments where, as local physicochemical conditions change under abiotic and biotic processes, microbial communities succeed one another. Studies of early colonisation in newly formed lava tubes created by the 2021 Tajogaite eruption on La Palma have already inferred an early development of nitrogen cycling. Evidence of an active nitrogen cycle on Mars raises the possibility of a biotic role in nitrogen transformations on the red planet, highlighting the importance of studying the nitrogen biogeochemical cycle at different stages of ecosystem development. As on Earth, Martian lava tubes may provide sheltered environments protected from radiation and characterised by relatively stable conditions, making them promising sites for biosignature preservation. Terrestrial lava tubes are thus valuable analogues to explore how Martian subterranean environments could support nitrogen‑cycling processes, preserve biosignatures and serve as testbeds for future exploration technologies.

However, despite evidence of nitrogen genes in lava tubes, no work has examined how these functions change with ecosystem maturity. Here, we investigate how nitrogen- related microbial functions evolve along a lava tube chronosequence.

Sediments, microbial colonies, mineral crusts, and speleothems from 10 lava tubes on La Palma, spanning ages from 4 years to ~2 million years, were collected. DNA was extracted using protocols optimised for low‑biomass, and microbial communities characterised through Illumina Miseq sequencing of the V3–V4 region of the 16S rRNA gene and MinION sequencing of the full-length 16S rRNA gene. Community composition and diversity were then analysed, and potential functions inferred with FAPROTAX. To investigate the genetic potential for nitrogen transformations, we performed PCR on a subset of samples targeting nifH (nitrogen fixation potential), amoA (nitrification potential), and nirK, nirS and nosZ (denitrification/N₂O sink potential). Furthermore, high‑resolution 3D models of the lava tubes were created, providing spatial and geomorphological context, relevant to the interpretation of nitrogen-cycling patterns and to support the geoconservation of these environments. Then, the biological results and metadata were combined to evaluate how nitrogen cycling potential evolves with ecosystem maturity and relates to environmental parameters.

Ongoing analyses, together with previous studies, indicate that recently formed lava tubes have lower DNA yields than more mature ones, reflecting harsher and more oligotrophic conditions. Furthermore, we hypothesise that, as lava tubes age and we have more mineral and nutrient inputs from water infiltration and mineral weathering, including fixed nitrogen, microbial communities will have more diverse nitrogen cycling metabolisms, as nitrogen fixation is energetically expensive compared to other nitrogen transformations.

This work stresses the importance of nitrogen fixation in sustaining microbial communities under oligotrophy, it advances our understanding of the biogenic potential of subsurface nitrogen cycling and the value of technologies and robotic systems for future terrestrial and planetary exploration. Future work will combine qPCR and cultivation to validate active nitrogen transformations, isolate key nitrogen‑cycling taxa and support biosample recovery technologies.

Acknowledgements: The Spanish Ministry of Science, Innovation and University from the Spanish State Agency (MICIU/AEI/10.13039/501100011033) is acknowledged for funding the HERMES project (ref. PID2024-162087NB-C21). FCT PhD grant M. Grilo (2025.04772.BD).