Metabolically diverse microorganisms mediating hydrocarbon cycling in the subseafloor sediment of the Challenger Deep

Hadal subseafloor sediments host abundant and active microbial biosphere with considerable heterotrophic activity. However, carbon and nutrient cycling processes and mechanisms driven by hadal subsurface microorganisms remain poorly understood. Using culture-dependent and culture-independent methods, we characterized the diversity, metabolism, and vertical dynamics of hydrocarbon-degrading (HYD) bacteria in a subsurface sediment core (MT20-750, ~750 cm below seafloor [cmbsf]) collected from the Challenger Deep (10,816 m below sea level) in the Mariana Trench. The sediment core contained high concentrations of mid- and long-chain n-alkanes (310-8,724 ng/g), although no 18 n-alkanes at detectable levels were present, in contrast to proximal hadal seawater samples where these compounds were highly abundant. Metagenomic analysis identified diverse genes for aerobic and anaerobic degradation of n-alkanes and aromatic compounds, distributed across a wide range of taxa, dominated by Chloroflexota, Planctomycetes, Proteobacteria, and Actinobacteria, alongside six novel HYD phyla. Metabolic reconstruction of 120 HYD metagenome-assembled genomes (MAGs) suggests distinct hydrocarbon degradation preferences and metabolic strategies among different bacterial groups. Additionally, hadal bacterial isolates from Proteobacteria, Actinobacteria, and Firmicutes were able to degrade n-alkanes (C_18-36), with Dietzia maris HXX048 removing ~50% of n-eicosane within 45 days under 5°C and 50 MPa. Predicted substrate binding of C₁₀ and C₂₀ with AhyA and heterologous expression of almA in Chloroflexota genomes supported their degradative capacity under anaerobic and aerobic conditions. The detection of putative hydrocarbon synthesis genes, specifically oleBC and oleC, suggests that hadal heterotrophic microorganisms may synthesize hydrocarbons. These findings provide evidence for microbial hydrocarbon production and thereby support a previously unrecognized sedimentary hydrocarbon cycle.IMPORTANCEOur findings suggest that hydrocarbon degradation may play an important role in organic matter decomposition and carbon cycling in the hadal subseafloor. This degradation capacity is likely distributed through diverse metabolic pathways across a wide range of phylogenetic taxa. The detection of genes likely encoding enzymes involved in aerobic and anaerobic hydrocarbon degradation, as well as the identification of novel hydrocarbon-degrading (HYD) phyla, highlights the complexity and significance of microbial processes in hadal subsurface sediment. The widespread distribution of hydrocarbon degradation capacity in different hadal sediments suggests hydrocarbons as a potential carbon source sustaining microbial life in this extreme environment. Moreover, the presence of genes associated with hydrocarbon synthesis suggests that hadal sediment microbes possess the genetic potential for both degrading and producing hydrocarbons, pointing to a dynamic and multifaceted hydrocarbon cycle within hadal subsurface sediment.