Microdroplet-Enabled Co-Cultivation and Characterization of MicrobialCommunities.

Microbial Community;Co-cultivation;Chemical Engineering;Engineering;Chemical Engineering

Park, Ji HyangYoung, Vincent Bensan ;

University of Michigan

关键词: Microbial Community;    Co-cultivation;    Chemical Engineering;    Engineering;    Chemical Engineering;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/91564/pjhyang_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

The majority of existing microbial species, in particular bacteria living in synergisticcommunities, have not been cultured in the laboratory. One important reasonbehind this unculturability;; is that conventional laboratory cultivation is aimed atpure cultures of individual species. The objective of this dissertation is to developmicrouidic co-cultivation platforms to expand the repertoire of cultivable speciesfrom natural microbial communities and to characterize co-cultivated communities.We first aimed to make use of highly parallel micro-droplets to co-cultivate symbioticmicrobial communities. We fabricated a microuidic device that could readilyencapsulate and co-cultivate subsets of a community, using aqueous droplets dispersedin a continuous oil phase. To demonstrate the effectiveness of this approach in discoveringsynergistic interactions among microbes, we tested it with a synthetic modelsystem consisting of cross-feeding E. coli mutants. This technology is being extendedand applied to the investigation of drug-producing natural microbiota isolated fromtunicates.We next combined droplet co-cultivation with oxygen gradient generation to provideboth the optimal oxygen concentration and the environment for microbial interxactions. Our device was composed of two glass layers with uid channels separatedby a 50-m-thick PDMS membrane. A linear oxygen gradient established in the gaschannel was successfully transferred to droplets in the liquid channel. A murine fecalmicrobial sample, of which the bacteria lived with limited oxygen concentrationin their native environment, was cultivated and different species were enriched inchambers 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 millionsof droplets using a microcentrifuge tube, and have combined it with a microuidicdevice for automated droplet sorting. Automated sorting is performed hydrodynamicallybased on analysis of uorescent droplet images representing cell density aftercultivation.This dissertation demonstrates that droplet-enabled co-cultivation can effectivelydecompose complex microbial communities into subsets of symbiotic members andthus facilitate the elucidation of underlying microbial interactions. In addition, automateddroplet sorting can be exploited for engineering purposes such as ultrahighthroughputscreening of microbial strain libraries.