Microfluidic-based high-throughput isolation enhances the recovery of novel strains and diversity from Arctic soil microbiome.

Abstract

Microbial cultivation remains essential for understanding the physiology, ecology, and biotechnological potential of environmental microbes, yet conventional plate-based methods (CPM) recover only a minute fraction of the environmental microbiome. Polar regions, particularly Arctic soils, represent unique reservoirs of "microbial dark matter" that remain challenging to cultivate, owing to oligotrophic conditions, low temperatures, and freeze-thaw cycles that impose severe physiological constraints on microbial growth. Here, we report the first systematic application of microfluidic droplet technology (MDT) to Arctic active-layer soil microbiota and benchmark its performance against CPM using identical starting cell numbers, R2A medium, and incubation at 15°C. MDT achieved 6.5- to 8.1-fold higher recovery rates than CPM and improved isolation throughput by >180-fold. Near-full-length 16S rRNA gene sequencing (PacBio) revealed that MDT recovered significantly higher taxonomic richness across all taxonomic levels, with 256 genera detected in the high-cell-input group (DropAS_H) versus 211 in the corresponding plate group (PlateAS_H). Notably, MDT yielded a more even community distribution, significantly reducing the dominance of fast-growing copiotrophs, such as Pseudomonas and Flavobacterium. Moreover, approximately 50% of the sequences from MDT were affiliated with potential novel species (<98.65% identity to type strains), and 27% with potential novel genera (<95% identity). Strain verification by Sanger sequencing confirmed 12 of 17 isolates as candidate novel species, among which one strain represented a potential novel genus within Devosiaceae. This study demonstrates that MDT is a powerful platform for accessing the uncultured majority of polar soil microbiota and establishes a pipeline for high-throughput isolation of novel cold-adapted bacteria.IMPORTANCEArctic soils harbor a vast reservoir of microbial diversity that remains largely inaccessible due to the extreme oligotrophic conditions and low temperatures characteristic of polar environments, leading to slow growth and extended lag phases in most microbes. Conventional plate-based methods (CPM) inherently favor fast-growing copiotrophs while suppressing rare or slow-growing lineages. Here, we demonstrate that microfluidic droplet technology (MDT) overcomes these fundamental constraints, representing its first systematic application to polar microbiology. By physically isolating individual cells into microliter-scale bioreactors, MDT provides independent microenvironments that allow slow-growing and oligotrophic taxa to proliferate without being outcompeted by fast-growing copiotrophs in bulk cultures. The water-in-oil emulsion format further enables extended low-temperature incubation without evaporative loss or airborne fungal contamination, issues that frequently compromise long-term plate-based cultivation of Arctic samples. Relative to CPM, MDT increased recovery rates by over 6-fold and isolation throughput by 180-fold, while markedly enhancing both taxonomic richness and evenness. Exclusively recovered by MDT, the oligotrophic genus Caulobacter and numerous cold-adapted genera underscore that MDT accesses physiologically distinct fractions of the cryospheric microbiome. Furthermore, the integration of near-full-length 16S rRNA gene sequencing with MDT cultivation assessment provided substantially improved phylogenetic resolution for species-level identification and novel taxon delineation. Collectively, these findings establish MDT as a transformative platform for cryospheric culturomics, accelerating the construction of comprehensive polar strain collections essential for understanding cold-adaptation mechanisms and exploiting the biotechnological potential of Earth's frozen microbiomes.