Cultivation strategies for prokaryotes from extreme environments.

The great majority of microorganisms are as-yet-uncultivated, mostly found in extreme environments. High-throughput sequencing provides data-rich genomes from single-cell and metagenomic techniques, which has enabled researchers to obtain a glimpse of the unexpected genetic diversity of "microbial dark matter." However, cultivating microorganisms from extreme environments remains essential for dissecting and utilizing the functions of extremophiles. Here, we provide a straightforward protocol for efficiently isolating prokaryotic microorganisms from different extreme habitats (thermal, xeric, saline, alkaline, acidic, and cryogenic environments), which was established through previous successful work and our long-term experience in extremophile resource mining. We propose common processes for extremophile isolation at first and then summarize multiple cultivation strategies for recovering prokaryotic microorganisms from extreme environments and meanwhile provide specific isolation tips that are always overlooked but important. Furthermore, we propose the use of multi-omics-guided microbial cultivation approaches for culturing these as-yet-uncultivated microorganisms and two examples are provided to introduce how these approaches work. In summary, the protocol allows researchers to significantly improve the isolation efficiency of pure cultures and novel taxa, which therefore paves the way for the protection and utilization of microbial resources from extreme environments.