Droplet microfluidic multistep platform for high-throughput active selection of cellulolytic microorganisms

• Development of an integrated picoinjection and sorting device for multi-operation microfluidic protocol. • Establishment of a droplet-based colorimetric assay for cellulose quantification. • Novel absorbance-based screening for selective enrichment of cellulolytic bacteria. Classical enrichment methods for microorganisms rely on growth in selective media, but such practices are relatively expensive, low-throughput, and may result in a biased representation of taxa. Alternatively, microorganisms can be cultivated in thousands of picoliter droplets of equal volume, with the most efficient strains selected through quantitative assays at unprecedented ultrahigh throughput. Here, we present a novel high-throughput microfluidic technology for the characterization of cellulolytic microbial communities at the single-cell level. Individual microbial cells are encapsulated in picoliter droplets for clonal cultivation, after which a colorimetric assay using Congo red is applied to identify positive droplets containing cellulose-degrading strains. These positive droplets are then actively sorted at high throughput using absorbance-activated droplet sorting. The critical component of the enrichment assay we propose is the integrated picoinjection and sorting device, which expands the range of available droplet-based enrichment methods. The platform introduces a new design that enables buffer picoinjection required for the assay, followed by droplet sorting on a millisecond timescale. A key strength of this module is the execution of multiple microfluidic operations within a single device. Achieving such multi-step processing is challenging, as it requires precise droplet manipulation to prevent droplet loss, unintended merging, or cross-contamination. We addressed these challenges with a chip architecture that allowed accurate and swift delivery of reagents without compromising downstream detection and sorting of droplets thus providing a compact and versatile system for absorbance-based droplet protocols.