High-density microdroplet cultivation reveals the essential role of microbial interactions in the growth of...

The significance of microbial interactions in nature is not well understood because of technical limitations in observing how individual microbial cells begin to grow within complex microbial community. 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 cell density (>10⁸ 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 is the key feature 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. The 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, revealing the crucial role of microbial interactions in the growth of environmental microorganisms and contributing to solving the issue of microbial uncultivability.

The overwhelming majority of environmental microbes remain uncultured, limiting our understanding of their physiology and ecological roles. Although microbial interactions have been predicted as one of the key factors for the growth of uncultivable microbial types, the effect of these interactions on cultivability remains poorly understood. In this study, we developed a new droplet-based co-cultivation approach that promotes microbial interactions while maintaining pure cultures and enables growth tracking at the single-cell level. This method significantly improved cultivability (approximately 10 times), including the growth of taxa that are difficult to cultivate. Direct observation of microbial growth in the community using this method clearly demonstrated that microbial interactions are essential for the growth of diverse microbial types. These findings underscore the importance of microbial interactions in cultivation and offer a basis for radically expanding microbial bioresources, manipulating microbial communities, and exploring previously unrecognized microbial interactions.