Description
Background and aims: The selection of cellulolytic microorganisms, together with the ability to bridge the genotype-to-phenotype gap, is essential for biotechnological applications such as biofuel production, lignocellulosic waste valorisation, and soil bioremediation. Conventional screening methods based on cultivation on selective solid media are typically time-consuming, low-throughput, and biased towards fast-growing taxa, thereby overlooking many valuable candidates such as slow-growing or rare strains. To overcome these limitations, we encapsulated single cells in picolitre droplets, each functioning as an individual bioreactor for clonal growth, and subsequently screened them using the PicoSorter, a novel microfluidic platform enabling high-throughput and simultaneous microfluidic operations for the selection of cellulose-degrading cultures.
Methods: The PicoSorter integrates picoinjection with absorbance-activated droplet sorting (AADS) on a single microfluidic chip, enabling the execution of multistep optical assays in droplet format. Individual microbial cells are encapsulated in 50 pL droplets containing carboxymethylcellulose (CMC) and Congo red dye, which binds to cellulose polymers. Following off-chip incubation and injection of MOPS/NaCl buffer into the droplets, the microcultures were sorted based on their transmittance signal, as droplets encapsulating cellulose-degrading strains exhibited measurable absorbance changes.
Results: The PicoSorter integrates several key components within a compact double-layer design, including a droplet reinjection chamber, spacing channels, a buffer picoinjection section with a first set of electrodes, a serpentine mixing region, channels for high–refractive index oil, and a sorting section equipped with a bias oil channel and a second set of electrodes. Altogether, these features enable accurate transmittance detection from droplets and ensure optimal sorting performance.
Initial tests to quantitatively assess CMC concentrations revealed a correlation between droplet transmittance signals and CMC content. Validation using a binary microbial community composed of Cellulosimicrobium cellulans (cellulolytic) and Escherichia coli (non-cellulolytic) demonstrated successful detection of microbial cellulolytic activity after dynamic incubation in Bushnell Haas droplets supplemented with CMC and Congo red. The PicoSorter achieved sorting rates of up to 0.6 kHz and high enrichment factors, indicating that our approach effectively enriches cellulolytic microorganisms.
Conclusions: The PicoSorter addresses the limitations of traditional microbiological isolation protocols, opening new opportunities for both academic research and biotechnological applications. Its versatility allows for broad applicability across different microfluidic assays. Beyond the proposed optical assay, the PicoSorter can also be used to detect fast reactions involving substrates that leak from droplets into the oil phase or between droplets. This method represents significant progress in microbial screening and enrichment, and the developed device can be applied across diverse areas of modern environmental microbiology, including biofuel production, bioremediation, and sustainable industrial processes.
Keywords: Droplet microfluidics, high-throughput screening, absorbance-activated droplet sorting, cellulolytic microorganisms.