A novel anaerobic n-Hexadecane hydroxylation pathway in Thermalkanevorax longiformis gen. nov., sp. nov. isolated from a deep-sea hydrothermal vent

Hydrothermal systems contain a wide spectrum of alkanes derived from biological sources and geological processes. However, despite their widespread occurrence in hydrothermal environments, the capacity and mechanisms of anaerobic alkane degradation by members of the phylum Bacillota remain poorly understood. In this study, we isolated a bacterial strain (designated strain L01) from the Lost City hydrothermal field (LCHF) by adding crude oil for enrichment culture. Strain L01 can grow anaerobically at 55 {degrees}C and pH 7.0 in medium supplemented with n-hexadecane, indicating its capability for anaerobic alkane degradation. Phylogenetic analysis based on the 16S rRNA gene sequence (93.11% similarity to its closest described relative) and average amino acid identity (AAI, 66.27-66.58%) revealed that strain L01 represents a novel genus within the family Symbiobacteriaceae, for which the name Thermalkanevorax longiformis gen. nov., sp. nov. is proposed. Cells of strain L01 are elongated rods measuring approximately 10-20 m in length and [~]0.2 m in diameter. Integrated genomic, transcriptomic, and metabolomic analyses revealed a previously unrecognized anaerobic n-hexadecane utilization pathway, involving initial alkane activation by AhyA homologous enzymes followed by stepwise oxidation and {beta}-oxidation. Together, these findings provide new insights into anaerobic alkane metabolism by hydrothermal bacteria, expanding current understanding of microbial hydrocarbon degradation in extreme marine environments. IMPORTANCEAlthough alkanes have be reported degradation by microorganisms through both aerobic and anaerobic metabolic pathways in hydrothermal ecosystems, the mechanisms underlying anaerobic alkane degradation remain incompletely understood, and the metabolic strategies employed by hydrothermal microorganisms are still poorly characterized. In this study, a novel genus bacterium Thermalkanevorax longiformis within the Symbiobacteriaceae family was isolated from the LCHF and identified an unrecognized C1-position n-hexadecane hydroxylation of AhyA homologous proteins at the anaerobic condition. These findings not only provide fundamental insights into the metabolic strategies of hydrothermal bacteria but also expand our understanding of microbial hydrocarbon degradation in extreme marine environments, offering a potential genomic resource for biotechnological applications in hydrocarbon remediation.