Microfluidic enrichment of proteolytic microbial consortia from sewage sludge.

Proteolytic microbial consortia are key drivers of protein hydrolysis in complex organic substrates. In anaerobic digestion systems, such as those used for biogas production from sewage sludge, this process constitutes the initial and rate-limiting step. Despite their importance, proteolytic microorganisms remain poorly characterized due to the complexity of environmental microbiomes and the limitations of conventional cultivation and screening methods. Here, we present a label-free microfluidic protocol for the high-throughput cultivation and characterization of proteolytic microorganisms. Single microbial cells are encapsulated in gelatin droplets and grown clonally, where proteolytic activity is detected through image-based analysis of droplet shape changes. Enrichment of individual proteolytic cultures is achieved using a separate microfluidic device that enables passive droplet sorting. Taxonomic characterization of sorted droplets by 16S rRNA gene sequencing revealed a fivefold higher number of amplicon sequence variants (ASVs), and a more diverse array of proteolytic strains were recovered compared with conventional skim milk agar screening (SMA). Taken together, this microfluidic workflow allows accurate and fast enrichment of proteolytic strains. Our approach advances the understanding of proteolytic communities in sewage sludge and opens new opportunities for targeted microbial recovery in waste-to-energy applications. IMPORTANCE: Proteolytic microorganisms drive the initial and rate-limiting step of protein degradation in anaerobic digestion systems, such as sewage sludge biogas production, yet their diversity and function remain poorly characterized due to the limitations of conventional cultivation methods. We present a label-free droplet microfluidic workflow that enables high-throughput, single-cell cultivation, functional screening, and selective enrichment of proteolytic microbes directly from complex communities. This approach substantially improves the recovery and diversity of proteolytic strains compared with traditional assays, providing a powerful tool to study hydrolytic consortia and to enhance microbial discovery for waste-to-energy and other biotechnological applications.