Oil Composition Determines Microbial Preference for Carbon Source Between Hydrocarbons and Necromass in Water Droplets Within Crude Shale Oil

Water droplets entrapped in crude oil have recently been recognized as unexpected but metabolically active microbial habitats in shale oil reservoirs. However, the key environmental drivers that shape the community composition and metabolic profiles of these droplet microorganisms remain poorly understood. Here, we investigated three shale oil samples from Jiangsu oilfield, all of which contained abundant water droplets (>105 droplets/mL oil) of ancient formation water origin. Integrated microscopy, metagenomics and metabolomics revealed that the physicochemical properties of the enclosing oil, primarily API gravity and viscosity, are associated with droplet size and may influence the droplet microbiomes. These factors appear to jointly drive a fundamental metabolic dichotomy, where in light oil with low-salinity water (LOLS), communities are enriched with hydrocarbon degraders together with molecular signatures of active petroleum hydrocarbon metabolism. In more viscous, light-to-medium oil with high-salinity water (LMOHS), however, communities shift towards necromass recycling and strong genetic adaptations to osmotic stress. Our findings demonstrate that microbial life in water droplets enclosed in shale oil is widely found in the shale oil reservoirs examined, and selected by carbon quality and environmental pressure. This study provides a mechanistic framework for understanding microbial ecology and biogeochemical processes in shale oil reservoirs and offers insights for microbial enhanced oil recovery in heavy oil reservoirs.