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 bead-in-droplet biofilm cultivation 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. Insight BoxBiofilms are an important yet understudied form of microbial growth. In this study, we developed bead-in-droplet biofilm cultivation (BiDBiC), a novel ultra-high-throughput workflow to culture biofilms. The droplets act as massively parallelized miniature bioreactors, with co-encapsulated plastic beads providing a surface for cell attachment and growth. Using a well-characterized biofilm former, Stenotrophomonas maltophilia, as well as a drinking water biofilm isolate, Sphingopyxis sp. OPL5, we demonstrated robust adherent cell growth in droplets. We additionally efficiently separated beads from planktonic cells, enabling targeted molecular analysis and outgrowth of adherent cells. Adapting and extending BiDBiC could facilitate the study of numerous complex biofilm systems, such as diverse isolates or combinatorial subcommunities of microbiomes, to observe their phenotypes and probe underlying mechanisms.