Unrevealed prokaryotic diversity in a peridotite massif in an underground mine of New Caledonia

• First characterization of deep prokaryotic diversity in the Tiebaghi peridotite massif (New Caledonia). • Presence of biogenic methane detected, even at concentrations of H 2 below detection limits. • Dioxygen enables methanogen/methanotroph coupling, crucial for ecosystem structure and sustainability. • Taxonomic diversity differs significantly between biofilm and water samples, with distinct functional roles. The microbial diversity in an abandoned mine accessing the Tiebaghi peridotite massif in New Caledonia was studied. The site allowed sampling of alkaline water (pH 8.9-9.9) flowing through fractures, with no contamination from the atmosphere, ensuring optimal gas recovery. Two sampling campaigns collected gas samples, revealing variable gas flux. At one site, no bubbling was observed. At the other site, methane (0.11-1.2 mol%), CO 2 (0.054-0.08 mol%), and O 2 (7.5-18.3 mol%) were present, while H 2 was below detection. Methane showed a biogenic δ 13 C signature of -90‰ vs. vPDB (Vienna Pee dee Belemnite). The physico-chemistry indicated an oligotrophic environment with recent oxic meteoric water. Microbial biofilms, formed by microorganisms in the formation waters, included methanogenic archaea mainly from Methanobacteriaceae , likely responsible for methane production. In this oligotrophic environment, oxic waters supported methanotrophs ( Methylococcaceae ) and methylotrophs ( Methylophilaceae ), which may structure community dynamics. The presence of nitrogen-cycle families such as Nitrosococcaceae and Rhodocyclaceae raises questions, given the apparent depletion of reduced nitrogen compounds. The absence of H 2 suggests it may be consumed by hydrogenotrophic prokaryotes ( Methanobacteriaceae, Comamonadaceae ), reducing detectable levels. This study is the first to detail microbial diversity in the Tiebaghi massif, offering new insights into peridotite-associated microbial communities.