A diffusion-based integrative approach for culturing previously uncultured bacteria from marine sediments

Traditional cultivation methods with defined growth media can only isolate and cultivate a small number of microbes. However, much higher microbial diversity has been detected by cultivation-independent tools from a range of natural ecosystems. These represent a large unexplored pool of potentially novel taxa. In this study, a diffusion-based integrative cultivation approach (DICA) was developed to efficiently isolate novel taxonomic candidates from marine sediment. DICA combined a newly designed diffusion-based apparatus called a "microbial aquarium" with modified low-nutrient media. To determine the efficiency of DICA, cultivation results were compared with traditional cultivation approach (TCA). Both cultivation approaches resulted in the isolation of numerous representatives from the phyla Pseudomonadota, Actinomycetota, Bacteroidota, and Bacillota. However, the newly developed DICA also led to the successful cultivation of species from rarely cultivated phyla such as Verrucomicrobiota and Balneolota. Based on 16S rRNA analyses, the application of DICA resulted in the successful cultivation of 115 previously uncultured taxa out of a total of 196 isolates. Among these, 39 were identified at the genus level and 4 at the family level, showcasing a novelty ratio of 58%. Conversely, the TCA cultivated 12% (20/165) of novel isolates, with all at species level only. The isolated microbial diversity showed that species recovered by DICA belong to 12 different classes, twice the number produced by TCA. Overall, these results demonstrate that the newly designed DICA produces a high recovery of diverse and previously uncultured bacteria.