BiDBiC: A novel ultra-high-throughput pipeline for Bead-in-Droplet Biofilm Cultivation and Characterization

Biofilms are a form of microbial growth consisting of cells, often attached to a surface, embedded in a structured 3D extracellular matrix that confers important emergent properties such as increased resistance to physical removal and antimicrobials. Despite the importance of biofilms to a variety of systems and despite increasing attention from both the public and private sectors, high-throughput approaches to study them are scarce, limiting investigations of complex mechanisms critical for the structure and function of biofilms, such as interactions in multispecies communities. We thus developed a novel workflow to grow and analyze bacterial cells adhered to plastic beads encapsulated within highly parallel nanoliter-scale water-in-oil microfluidic droplets. We term this pipeline for b ead-in- d roplet bi ofilm c ultivation and characterization BiDBiC. To benchmark BiDBiC, we utilized a well-characterized biofilm former, Stenotrophomonas maltophilia , as well as a poorly studied drinking water biofilm isolate, Sphingopyxis sp. OPL5. Each bacterium exhibited strong adherent growth when co-encapsulated with polystyrene beads in droplets. Furthermore, we retrieved beads from the droplets and removed planktonic cells, enabling focused analysis of adhered cells. From bead-associated biomass, we extracted DNA and RNA for molecular analysis and recovered viable cells for subculturing. We conclude with a discussion of further development of the platform as well as suggestions for microbial biofilm systems that may benefit from ultra-high-throughput droplet-enabled cultivation and analysis.