Biology subjects

Kindaichi, T.

Publications and source records attributed to Kindaichi, T..

3 recordsLinked to original sources

bioRxiv2025

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 107 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 (>108 cells/mL) but maintains pure cultures of each growth unit. Millions of hydrogel particles (10-30 {micro}m 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. IMPORTANCEThe 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.

microbiology↗

bioRxiv2023

Unique episymbiotic relationship between Gracilibacteria and Zoogloea in activated sludge flocs in a municipal wastewater treatment plant

Among the various bacteria present in activated sludge, uncultivated Patescibacteria (also known as the Candidate Phyla Radiation/CPR superphylum) are ubiquitous at the class or phylum level. Patescibacteria have a highly restricted metabolic capacity and are thought to be episymbiotic/endosymbiotic or predatory. However, only a limited number of Patescibacteria and their hosts have been identified. Therefore, many Patescibacteria have not been (co-)cultured and identified by fluorescence in situ hybridization (FISH) or electron microscopy. Little is known about the morphology, metabolic potential, and hosts of Gracilibacteria (formerly GN02 or BD1-5) which belong to Patescibacteria. In our previous study, we confirmed the presence of Gracilibacteria in activated sludge and successfully recovered its high-quality genome. In this study, we designed new probes to visualize members of Gracilibacteria in activated sludge and identified its host using FISH. The FISH observations revealed that Gracilibacteria, which formed loosely associated clusters, were located within dense clusters of Zoogloea, which were dominant in the activated sludge. The metagenome-assembled genomes (MAGs) of Zoogloea possessed genes related to extracellular polymeric substance (EPS) biosynthesis, floc formation, and nutrient removal, including a polyhydroxyalkanoate (PHA) accumulation pathway. The MAGs of Gracilibacteria possessed genes associated with type IV pili, competence protein EC (ComEC), and PHA degradation, which suggests that they have a Zoogloea-dependent lifestyle in activated sludge flocs. These findings clearly indicate a new symbiotic relationship between Gracilibacteria and Zoogloea, and to the best of our knowledge, this is the first study to show this interaction.

microbiology↗

bioRxiv2019

Cultivating in situ triggers growth of previously uncultivated microorganisms via a growth initiation factor in nature

Most microorganisms resist cultivation under standard laboratory conditions. On the other hand, to cultivate microbes in a membrane-bound device incubated in nature (in situ cultivation) is an effective approach. In the present study, we applied in situ cultivation to isolate diverse previously uncultivated marine sponge-associated microbes and comparatively analyzed this methods efficiencies with those of the conventional method. Then, we attempted to clarify the key and unknown mechanism of in situ cultivation by focusing on growth triggering via growth initiation factor. We hypothesized that majority of environmental microorganisms are in nongrowing state and requiring \"growth initiation factor\" for the recovery and that can be provided from environments. Consequently, significantly more novel and diverse microbial types were isolated via in situ cultivation than by standard direct plating (SDP). Next, the effect of the sponge extract on starvation recovery was compared between strains derived from in situ and SDP cultivation. Adding small amounts of the sponge extracts to the medium elevated the colony-formation efficiencies of the in situ strains at the starvation recovery step, while it showed no positive effect on that of SDP strains. Conversely, specific growth rates or carrying capacities of all tested strains were not positively affected. These results indicate that, 1) the sponge extract contains chemical compounds that facilitate starvation recovery, these substances selectively worked on the in situ strains, and 2) growth initiation factor in the sponge extract did not continuously promote growth activity but worked as triggers for regrowth (resuscitation from dormancy).\n\nImportanceMost microbial species resist cultivation under laboratory condition. This is critical impediment for both academic and applied microbiology, and thus clarification of the mechanism of microbial uncultivability is highly demanded. Several evidences have been reported that to cultivate microbes in a membrane-bound device incubated in nature (in situ cultivation) is an effective approach. However, the mechanism behind this approach has not been clarified. The present study shows the evidence that 1) initiating growth is a key for cultivating previously uncultivated microbes rather than simple growth promotion, and 2) growth initiation factor (signaling-like compounds) in natural environments stimulate microbial resuscitation from a nongrowing state. Since no study has focused on growth initiation for cultivation of previously uncultivated microorganisms, the discovery shown in the present study provides a new insight into microorganisms previously considered uncultivable and a microbial growth controlling system in nature.

microbiology↗