Microdroplet-enabled co-cultivation and characterization of microbial communities

The majority of existing microbial species, in particular bacteria living in synergistic communities, have not been cultured in the laboratory. One important reason behind this "unculturability" is that conventional laboratory cultivation is aimed at pure cultures of individual species. The objective of this dissertation is to develop microfluidic co-cultivation platforms to expand the repertoire of cultivable species from natural microbial communities and to characterize co-cultivated communities.;We first aimed to make use of highly parallel micro-droplets to co-cultivate symbiotic microbial communities. We fabricated a microfluidic device that could readily encapsulate and co-cultivate subsets of a community, using aqueous droplets dispersed in a continuous oil phase. To demonstrate the effectiveness of this approach in discovering synergistic interactions among microbes, we tested it with a synthetic model system consisting of cross-feeding E. coli mutants. This technology is being extended and applied to the investigation of drug-producing natural microbiota isolated from tunicates.;We next combined droplet co-cultivation with oxygen gradient generation to provide both the optimal oxygen concentration and the environment for microbial interactions. Our device was composed of two glass layers with fluid channels separated by a 50-mum-thick PDMS membrane. A linear oxygen gradient established in the gas channel was successfully transferred to droplets in the liquid channel. A murine fecal microbial sample, of which the bacteria lived with limited oxygen concentration in their native environment, was cultivated and different species were enriched in chambers featuring different oxygen conditions.;To further parallelize and automate the droplet-enabled co-cultivation technology, we have also developed a simple and robust method for incubation of millions of droplets using a microcentrifuge tube, and have combined it with a microfluidic device for automated droplet sorting. Automated sorting is performed hydrodynamically based on analysis of fluorescent droplet images representing cell density after cultivation.;This dissertation demonstrates that droplet-enabled co-cultivation can effectively decompose complex microbial communities into subsets of symbiotic members and thus facilitate the elucidation of underlying microbial interactions. In addition, automated droplet sorting can be exploited for engineering purposes such as ultrahigh-throughput screening of microbial strain libraries.