Accessing rare bacterial biosphere of soil through culturing: a comparative study of culture media effectiveness integrated with metataxonomics

Soil harbors a vast bacterial diversity, including a “rare biosphere” of low-abundance taxa increasingly recognized as crucial for ecosystem functioning. Traditional plate-based culturing can facilitate access to this fraction. Here, we investigated how culture medium type influences the recovery of rare soil bacteria. To do this, culture-independent bacterial diversity of agricultural, forest, and contaminated soils was compared with culture-dependent diversity on four media (TSA (tryptic soy agar), SEM (soil extract medium), R2A (Reasoner’s 2A), and 20-fold diluted R2A) using a high-throughput approach in which all colonies were collected and analyzed via 16S rRNA gene metabarcoding (metataxonomics). Across all media and soils, ~30% of the culture-independent taxa were recovered through culturing, of which ~40% were members of the rare community. Culturability of bacterial communities declined across soil types in the order: contaminated > agricultural > forest, but the community isolated from forest soil contained a higher proportion of rare taxa. SEM, characterized by its low nutrient content and chemical composition resembling that of soil, proved to be the most effective, yielding higher colony-forming unit counts, greater overall and rare richness, and recovering a more taxonomically diverse rare community. The rare community retrieved by SEM was dominated by non–spore-forming taxa, in contrast to the other media, including diluted R2A. Culturing revealed a unique fraction of culturable taxa, representing ~17% of the culture-independent community on average, likely members of the rare biosphere that escape detection in metabarcoding analyses. SEM was also the medium that best captured this fraction. Our study demonstrates that low-nutrient, soil-like media such as SEM are powerful tools for accessing and characterizing the rare soil biosphere, complementing metabarcoding and providing isolates to explore the ecological and functional significance of rare taxa.