Cell‐in‐Bead‐in‐Droplet Platform for pH‐Based Microfluidic Screening of Ureolytic Bacteria

Ureolytic microorganisms are central to microbially induced carbonate precipitation (MICP), a biotechnological process with applications in construction, environmental remediation, and wastewater treatment. Despite their potential, the discovery of robust, high-performing ureolytic strains is limited by the lack of assays that measure single-cell enzymatic activity in high-throughput platforms, such as droplet microfluidic devices. Although pH-based assays using urea offer a direct and label-free readout of urease activity, their implementation in droplet microfluidics is hindered by chemical crosstalk through diffusing molecules. Ammonia, the volatile product of ureolysis, spreads between droplets, making it difficult to detect droplets that host high-performing cells. To overcome this limitation, we have developed a 'cell-in-bead-in-droplet' (CiBiD) microfluidic platform that enables reliable detection of localized pH changes within individual cell-laden droplets. Single bacterial cells are first encapsulated in agarose beads to proliferate into microcolonies. The cell-laden beads are then re-encapsulated into droplets containing urea, a pH-sensitive fluorescent dye, and a buffer. By boosting the local enzymatic activity in the droplet while neutralizing diffusing ammonia with the buffer, the CiBiD approach circumvents diffusional crosstalk and enables robust detection of urease activity based on localized pH variations. Using a mock microbial consortium, our system achieved a 25-fold enrichment of active ureolytic strains after sorting 628 out of approximately 240,000 droplets in less than 30 min. To demonstrate its potential for the biopropection of functional microorganisms from natural microbiomes, the methodology was also successfully utilized to enrich ureolytic bacteria from environmental soil samples. Beyond local pH detection, the CiBiD concept may be applied to other challenging assays in media that operate at extreme pHs, involve high salt concentrations or are prone to undesirable dye interference. This makes CiBiD an attractive screening tool for high-throughput bioprospection and directed evolution of microorganisms.