Abstract: Although microbial isolation techniques have been developed for over a century, conventional methods often fail to capture the vast majority of environmental microbial diversity, largely because slow-growing bacteria are outcompeted by fast-growing taxa. To address this limitation, we developed a simple liquid-medium-dilution (LMD) method that physically isolates single cells in liquid microcultures using limiting dilution and the Poisson distribution, without requiring specialized equipment. Comparative analysis of 787 bacterial strains isolated from estuarine sediments of the Qiantang River, China, showed that LMD recovered 72 putative novel species (19.2%), a significantly higher proportion than the traditional-solid-culture (TSC) method (47 strains, 11.4%; P < 0.01). Importantly, LMD uniquely accessed rare and slow-growing lineages, including members of the phylum Verrucomicrobiota and, most notably, two novel species, Poseidonivirga lenticrescens gen. nov., sp. nov., and Poseidonivirga cretacea sp. nov. These strains show low 16S rRNA gene sequence similarities (90.37% and 90.51%) to known taxa and represent a novel order, Poseidonivirgales, within the class Alphaproteobacteria. Our findings demonstrate that the LMD method effectively minimizes competitive exclusion, enabling the cultivation of previously unculturable, slow-growing, and phylogenetically deep bacterial lineages using standard laboratory conditions.

Key words: bacterial diversity, competitive exclusion, cultivation, limiting dilution, polyphasic taxonomy, single-cell isolation, slow-growing bacteria