Biology subjects

Potenza, L.

Publications and source records attributed to Potenza, L..

4 recordsLinked to original sources

bioRxiv2026

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 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 gelatine 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 passive microfluidic device that enables droplet sorting. Taxonomic characterization of sorted droplets by 16S rRNA gene sequencing revealed a fivefold higher number of 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 contributes to a deeper understanding of proteolytic communities in sewage sludge and opens new opportunities for targeted microbial recovery in waste-to-energy applications. ImportanceProteolytic 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.

microbiology↗

bioRxiv2025

Droplet microfluidic PicoSorter for high throughput and active selection of cellulolytic microorganisms.

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 PicoSorter module, 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. Highlights- Development of a novel device (PicoSorter) capable of performing multiple microfluidic operations simultaneously. - Implementation of a colorimetric droplet-based assay for the detection of cellulose. - The proposed absorbance-based HTS enables active and efficient enrichment of cellulolytic bacteria.

microbiology↗

bioRxiv2025

Induced Droplet Ovalisation (IDO): Image-based microfluidic method for high-throughput and label-free characterization of microbial proteolytic strains from wastewater sludge.

Traditional bacterial isolation methods are costly, labour-intensive, and often fail to detect rare or slow-growing taxa. In contrast, droplet-based microfluidic assays have emerged as powerful alternatives. Here, we describe a novel, label-free microfluidic assay for screening microbial proteolytic activity based on oil-induced droplet ovalization. This method outperformed two traditional isolation techniques in terms of performance, time, and overall cost-efficiency. We present Induced Droplet Ovalisation (IDO): a droplet-based protocol that combines single-cell encapsulation with automated image analysis. Using a custom-made microfluidic device and bespoke image-processing scripts, our system efficiently detects microbial proteolytic activity by monitoring droplet deformability. This approach significantly reduces the time and resources required to study proteolytic consortia, offering an automated, easy-to-implement, and label-free alternative to conventional screening. Our device achieves high-throughput screening (HTS) at 0.2 kHz while significantly reducing the cultivation medium volume used compared to traditional methods. Furthermore, in-droplet microbial recovery is enhanced by two orders of magnitude, enabling the enumeration of significantly more colonies compared to traditional screenings. Beyond microbiological applications, this versatile platform serves as a powerful tool for studying hydrogel degradation and polymerization dynamics, extending its applicability across research and industry.

microbiology↗

bioRxiv2024

Passive droplet microfluidic platform for high-throughput screening of microbial proteolytic activity.

Traditional bacterial isolation methods are often costly, have limited throughput, and may not accurately reflect the true microbial community composition. Consequently, identifying rare or slow-growing taxa becomes challenging. Over the last decade, a new approach has been proposed to replace traditional flasks or multi-well plates with ultrahigh-throughput droplet microfluidic screening assays. In this study, we present a novel passive droplet-based method designed for isolating proteolytic microorganisms, which are crucial in various biotechnology industries. Following the encapsulation of single cells in gelatin microgel compartments and their subsequent clonal cultivation, microcultures are passively sorted at high throughput based on the deformability of droplets. Our novel chip design offers a 50-fold improvement in throughput compared to previously developed deformability-based droplet sorter. This method expands an array of droplet-based microbial enrichment assays and significantly reduces the time and resources required to isolate proteolytic bacteria strains.

bioengineering↗