(236j) Oxygen Gradient Construction for Co-Cultivation of Microbial Communities In Droplets

In nature, synergistic microbial communities play important roles in a wide spectrum of ecosystems. The natural habitats of these communities provide the best environment for their proliferation, including interactions between neighboring microbes and the optimal oxygen concentration. Oxygen is toxic to some microbes, and oxygen tolerance or preference varies from one microbial species to another. Providing various concentrations of oxygen, therefore, can greatly enhance the diversity of cultivated species in a community. Previously, we developed a microfluidic device for highly parallel co-cultivation of microbial communities in droplets and demonstrated its effectiveness in discovering synergistic interactions among microbes. In this work, we have extended this technology and developed a multilayered device for co-cultivation of microbes in droplets under various oxygen conditions ranging from 0% (anaerobic) to 21% (fully aerobic) of oxygen by constructing a gradient of oxygen concentration. Our device is composed of two glass channels separated by a 50-mm-thick PDMS membrane. Oxygen gradient was created through a tree-shaped channel mixing humidified nitrogen/oxygen gas flows and transferred through the porous PDMS membrane to the droplet-incubating liquid chambers. Linear and exponential gradients can be generated by designing appropriate geometry of the tree-shaped part of the gas channel and verified by measuring the phase change of fluorescence from a dye RTDP contained in droplets. As an initial test, we cultivate an artificial community constituted of several microbes with various oxygen preferences. Afterwards, natural microbiota such as tunicate microbiota associated with anticancer drug production, which has limited oxygen concentration in their original environment, will be investigated. Combined with the co-localization of symbiotic microbes in droplets, establishment of culture conditions mimicking natural habitats can expand the repertoire of cultivable microbes and also help to elucidate microbial interactions in a community.