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

Abstract The low cultivability of environmental bacteria has been widely acknowledged, but previous estimates focused on the proportion of cultivable cells (PCC) rather than cultivable taxa (PCT). Here we determined PCT through sequencing to profile bacterial taxa and genomes of the inoculum and culturomic agar plates for two types of environmental samples (soil and activated sludge). Results reveal that PCTs at the culturomics-level significantly exceed PCCs. Both amplicons and metagenomes indicate the presence of a large proportion of novel taxa in culturomics, albeit mostly at low relative abundances, consistently linked to the prevalence of microcolonies on plates. The distribution of novel taxa in culturomics guided the isolation of a strain representing a yet-uncultured class in the Bacteroidota. More importantly, with the definition of cultivable/uncultivable phenotype among environmental bacteria, we detected that cultivable taxa exhibit higher metabolic activity, under both in-situ conditions and laboratory treatment, as assessed by cellular rRNA content. Linking uncultivable phenotypes to genomic features facilitated the modification of culture media, and successfully enhanced the cultivable diversity of Burkholderiales from activated sludge samples. In conclusion, our study provides an experimental approach to reassess the cultivability of environmental bacteria and offers a promising path to optimize microbial cultivation.