Cultivating the uncultured: Harnessing the "sandwich agar plate" approach to isolate heme-dependent bacteria from marine sediment

In the classical microbial isolation technique, the isolation process inevitably destroys all microbial interactions and thus makes it difficult to culture the many microorganisms that rely on these interactions for survival. In this study, we designed a simple coculture technique named the "sandwich agar plate method," which maintains microbial interactions throughout the isolation and pure culture processes. The total yield of uncultured species in sandwich agar plates based on eight helper strains was almost 10-fold that of the control group. Many uncultured species displayed commensal lifestyles. Further study found that heme was the growth-promoting factor of some marine commensal bacteria. Subsequent genomic analysis revealed that heme auxotrophies were common in various biotopes and prevalent in many uncultured microbial taxa. Moreover, our study supported that the survival strategies of heme auxotrophy in different habitats varied considerably. These findings highlight that cocultivation based on the "sandwich agar plate method" could be developed and used to isolate more uncultured bacteria. Digging into uncultured microbes has been one of microbiology's most active research topics. In this study, we designed a simple and efficient coculture system to isolate uncultured microorganisms, and many uncultured commensal bacteria in marine sediment were successfully isolated. Our findings suggest that heme is a key growth factor mediating special microbial interactions and provides insight into the potential niche partitioning factors of different heme auxotrophy. Our approach constructs a model for exploring commensal microbes. This study also provides a new train of thought to test and mine the potential ecological functions of rare species.