Sequencing-guided re-estimation and promotion of cultivability for environmental bacteria

The low cultivability of environmental bacteria has been widely acknowledged, but most previous estimates focused on the proportion of cultivable cells rather than cultivable taxa. Here, we estimate the proportions of cultivable cells and cultivable taxa for two sample types (soil and activated sludge) using cell counting, 16S rRNA gene amplicon sequencing, metagenomics, and cultivation on agar plates under various conditions. We find that the proportion of cultivable taxa exceeds that of cultivable cells at the sample level. A large proportion of cultivable taxa are taxonomically novel but tend to be present at very low abundance on agar plates, forming microcolonies, and some of them cease to grow during subculture. Compared with uncultivable taxa (under the conditions used in our study), cultivatable taxa tend to display higher metabolic activity as inferred by measuring rRNA copies per cell. Finally, we use the generated taxonomic and genomic information as a guide to isolate a strain representing a yet-uncultured class within the Bacteroidota and to enhance the cultivable diversity of Burkholderiales from activated sludge.