Droplet microfluidics has emerged as a powerful tool for single-cell microbiology, enabling high-throughput cultivation, functional screening, and sequencing within picoliter to nanoliter compartments. Compared to conventional agar plates, droplet-based cultivation more efficiently recovers microbial diversity—including rare taxa—from environmental samples. Our previous work using a microfluidic streak plate (MSP) approach revealed that over 90% of operational taxonomic units (OTUs) captured in droplets belonged to the rare biosphere and were not detected on agar plates.
Building on this, we applied droplet-based single-cell cultivation under high hydrostatic pressure (up to 110 MPa) aboard a deep-sea research vessel to study microbial diversity in deep-sea water from the Southern Mid-Atlantic Ridge. Microbes were encapsulated in picoliter droplets, incubated under in situ pressure, and recovered by double emulsification for agar-based isolation. This approach yielded 70 species, including novel piezotolerant taxa such as Aequorivita, Psychrobacter, and Sphingopyxis. Multi-omics analyses revealed that pressure confinement enriched functions like osmoprotection, membrane stabilization, and quorum sensing.
These findings affirm droplet microfluidics as a transformative platform for accessing deep-sea microbial dark matter. Beyond high-pressure cultivation, we are also advancing fluorescence-activated droplet sorting for enzyme discovery and genome-targeted single-cell amplification and sequencing—expanding the microbial exploration toolkit across extreme, natural, and engineered ecosystems.
 

Droplet microfluidics,Single-cell cultivation,Deep-sea microbiome,High hydrostatic pressure,Microbial dark matter