Single-cell microliter droplet screening microfluidic system enables high-throughput isolation and cultivation of lignin-degrading bacteria from the termite gut.

Termite gut microbiome hosts diverse lignin-degrading bacteria, yet a substantial proportion remains uncultivated due to the complexity of their microbial interactions and growth requirements. In the current study, a novel single-cell microliter droplet screening microfluidic system (MISS Cell) was employed and compared with the conventional agar plate method for the high-throughput isolation and cultivation of lignin-degrading bacteria from the gut system of the higher termite Nasutitermes tiantongensis. The amplicon sequencing of 16S rRNA was conducted to assess the diversity of cultured bacteria. Compared to the conventional method, the MISS Cell system significantly improved the recovery of diverse ligninolytic bacteria, isolating 477 individual ligninolytic bacterial colonies, whereas the traditional method recovered only 73 colonies. A total of 97 operational taxonomic units (OTUs), including Acinetobacter sp. CIP_64.2, Advenella kashmirensis, and Staphylococcus sciuri, with 29 classified and 68 unclassified OTUs, were successfully obtained from both methods. The MISS Cell system yielded 16.7% more OTUs than the traditional agar plate approach. The bacterial isolates belonging to the phyla Proteobacteria, Firmicutes, Bacteroidota, and Actinobacteriota, comprising 23 families, 31 genera, and 54 species, were captured using both methods. Additionally, about 8.24% of the OTUs remained unclassified at the phylum level, underscoring the need for further taxonomic characterization of termite gut microbiota. These findings emphasize that the currently known repertoire of ligninolytic bacterial species is still incomplete and demonstrate the potential of the droplet-based MISS Cell system for uncovering novel microbes with lignin-degrading capabilities.