Cultivation of yet-to-be cultured microorganisms: advances, strategies, and prospects

More than 99% of microorganisms in the natural environment are not readily culturable using standard laboratory techniques. These microbes can be reservoirs of novel metabolites and biomolecules having pharmaceutical applications against bacterial infections, chronic diseases, and antibiotic resistance. Given this, our work is a comprehensive synthesis of recent advances in understanding, detection, and cultivation of "yet-to-be cultured" (YTBC) microbes. We highlight physiological traits that restrict their domestication under standard laboratory conditions. Some of the factors that may influence are their metabolic dormancy, specialized nutrient demands, siderophore-mediated iron acquisition, microbial signaling, and interspecies interactions. The review discusses various strategies, such as simulated natural environments, co-culture, and advanced bioreactor systems, which can be implemented to cultivate them. We reviewed recent metagenomic approaches and single-cell isolation methods, including label-based techniques (e.g. fluorescence in situ hybridization), label-free approaches such as Raman-activated cell sorting, and high-throughput tools like flow cytometry. We also examined culture-dependent approaches, including co-cultivation with helper strains with a special emphasis on bioreactor-based systems, diffusion chamber, hollow-fiber membrane chamber, high-throughput isolation chip (iChip), and encapsulation. Overall, this review provides a roadmap to unlock the biotechnological potential of YTBC microbes by outlining new technologies, methodological trends, and important knowledge gaps.