Microfluidic droplet cultivation under extreme pressure enables isolation and characterization of distinct deep-sea microbial dark matter

Deep-sea microorganisms comprise the Earth's largest and least explored microbiome, yet the vast majority remain uncultivated due to challenges of preserving in situ high hydrostatic pressure and preventing loss of viability and diversity during recovery, which limits our ability to explore their ecological functions and adaptive strategies. Here, we introduce DeepDrop, a microfluidics platform that enables high-throughput single-cell cultivation under pressures spanning the full ocean depth directly aboard research vessels, following direct colony formation via pipette-generated double emulsions. Applying to hadal samples, DeepDrop recovered >50% more microbial diversity than conventional high-pressure bulk cultivation, including rare taxa with streamlined genomes and distinctive genetic features associated with pressure adaptation. Combined metagenomic and transcriptomic analyses revealed that DeepDrop enriched pressure-adapted taxa carrying key stress-related genes and induced coordinated transcriptional reprogramming, characterized by upregulation of stress pathways and repression of motility. By integrating shipboard deployment, pressure-stable droplet cultivation, and efficient recovery, DeepDrop offers a powerful platform for accessing deep-sea microbial dark matter and illuminating microbial life strategies under extreme environmental constraints.