[Stability and viability optimization for filamentous microorganisms in droplet-based microfluidic screening]

Filamentous microorganisms-encompassing filamentous fungi and actinomycetes-are versatile cell factories for producing proteins and bioactive natural products, playing a critical role in the food and pharmaceutical industries. These microorganisms exhibit slow growth and complex morphological differentiation. Conventional screening methods for strain engineering are hindered by low throughput and an inability to resolve heterogeneity in strain cultivation and growth. Droplet-based microfluidics offers an attractive platform for high-throughput screening at the single-cell level, yet its utility is limited by poor droplet stability and low post-sorting viability. Given the aforementioned technical bottlenecks, this study aims to compare the compatibility between filamentous fungi and actinomycetes and droplet microfluidic technology, optimize post-sorting strain viability, and thereby establish a droplet microfluidic screening platform applicable to diverse filamentous microorganisms. Here, we systematically evaluated and optimized droplet-based microfluidic screening for filamentous fungi-Acremonium chrysogenum and two actinomycetes, Streptomyces lividans and Saccharopolyspora erythraea. Monodisperse spore-suspension preparation protocols and droplet incubation time were optimized for each organism, which revealed that actinomycete-generated droplets were significantly more stable than those of filamentous fungi, with optimal sorting windows dictated by strain-specific growth and morphological traits. Subsequently, we varied sorting voltage and recovery regimens (no treatment, demulsification, or demulsification followed by shaking incubation). Lower sorting voltage markedly improved viability, while demulsification effectively boosted viability. These targeted optimizations establish a practical workflow that converts droplet-based microfluidics into a robust tool for high-throughput screening of filamentous microorganisms.