High-Density Microdroplet Cultivation Reveals the Essential Role of Microbial Interactions in the Growth of Environmental Microbes

The significance of microbial interactions in nature is not well understood because of the limitations of techniques to assess microbial growth at the single-cell level under conditions of high cell density (more than 10 7 cells/mL). To address this limitation for further solution of microbial uncultivability, we developed a Gel Microdroplet Aggregate in Oil (GMD-agg) cultivation method. This method provides a high inoculum cell density (>10 8 cells/mL) but maintains pure cultures of each growth unit. Millions of hydrogel particles (10-30 µm in diameter) entrapping single cells with medium softly aggregated in oil (GMD-oil cultivation) is the key structure of this method. In this study, to assess the effect of microbial interactions on the cultivation of environmental microorganisms, soil and activated sludge samples were cultured, and colony formation and diversity during cultivation in GMDs were investigated. Results showed that the cultivation efficiency of GMD-agg was approximately 10 times higher than that of the method in which microbial interactions were canceled. In addition, the growth of taxonomies containing many uncultured microorganisms, such as Verrucomicrobia , Planctomycetia , Acidobacteria , and Vicinimibacteria , was observed. Furthermore, when isolated strains were co-cultured at high cell densities with either inter- or intra-species, we observed a higher cultivation efficiency than when microbial interactions were canceled. Rebuilding growth-promoting microbial interactions using isolated strains demonstrated that microbial interactions positively influence microbial growth. These findings indicate the effectiveness of this cultivation method, reveal the crucial role of microbial interactions in the growth of environmental microorganisms, and contribute to solving the issue of microbial uncultivability.