High-throughput anaerobic cultivation and profiling of porcine gut microbiota via a compact vibrating capillary-based droplet system

High-throughput cultivation and profiling are essential for elucidating the diversity, ecology, and functionality of gut microbiota. Although droplet microfluidics has emerged as a powerful tool in microbiology, its application to gut microbiota has been limited by complexities in anaerobic integration and operation. Here, we present a compact droplet system utilizing a simple vibrating sharp-tip capillary, capable of encapsulating and cultivating porcine colon microbiota in millions of droplets, with adjustable bacterial distributions through droplet size modulation. To investigate how varying complexities of interspecies interactions impacted the assembly and diversity of a gut microbial community, colon microbiota from a 21-day-old piglet was parallelly cultivated under three conditions: small droplets (Drop-S), large droplets (Drop-L), and bulk medium (Tube), corresponding to minimal, limited, and complex interspecies interactions, respectively. All three groups were incubated for 7 days, and colon microbiota without cultivation was collected as the Unculture group. To further identify specific gut bacteria associated with the biotransformation of a porcine-specific primary bile acid, hyocholic acid, the porcine colon microbiota was encapsulated in droplets with hyocholic acid and incubated for 7 days (HCA). Microbial communities from all five groups were retrieved via centrifugation and analyzed by quantitative full-length 16S rRNA gene sequencing.