Methanotrophy Under Extreme Alkalinity in a Serpentinizing System

Serpentinization produces hyperalkaline, H₂- and CH₄-rich fluids that support microbial life in extreme conditions and serve as analogs for ocean worlds such as Enceladus. While methane production in these systems has been well studied, methane consumption—especially under high pH—remains poorly understood. Here, we present isotopic, geochemical, and genomic evidence for hyperalkaliphilic (pH > 11) methanotrophy in the Samail Ophiolite in Oman. Using models that account for fluid mixing and gas exsolution, we identify δ¹³CH₄ enrichment that cannot be explained by abiotic processes alone. The enrichment of ¹³CH₄ co-occurs with methanotroph 16S rRNA gene sequences, particularly in fluids formed by mixing CH₄-rich, anoxic fluids with oxidant-rich surface waters. Shotgun metagenomics reveals a metagenome-assembled genome affiliated with Methylovulum, encoding a complete methane oxidation pathway, multiple carbon assimilation routes, and Na⁺/H⁺ antiporters—adaptations likely enabling growth above pH 11. Methanotroph diversity and abundance peak in mixed fluids but are suppressed at total ammonia nitrogen concentrations >20 μM. Anaerobic methane-oxidizing archaea (ANME) may also contribute to CH₄ oxidation in the deep subsurface. Our findings highlight the viability of methanotrophy under extreme alkaline conditions and provide a framework for interpreting δ¹³CH₄ signals in serpentinizing environments on Earth and beyond.