(180g) Micro-Droplet Enabled Co-Cultivation of Symbiotic Bacteria

In nature, most microbes live in synergistic communities as a way to adapt to and thrive in their environments, such as ocean, soil, and higher organisms as hosts. These microbial communities play important roles in a wide spectrum of ecosystems and form diverse interactions with one another and with their surroundings. Microbial interactions in natural microbiota are, in many cases, crucial for the sustenance of the communities, but these interactions remain largely unknown because of the inherent complexity and difficulties in laboratory cultivation. Most of previous works were based on genetic identification, while laboratory co-cultivation for elucidating microbial intercellular networks have been hardly investigated so far. In this work, we developed a simple microfluidic device for highly parallel co-cultivation of symbiotic microbial communities and demonstrated its effectiveness in discovering synergistic interactions among microbes. Using aqueous micro-droplets, which were serially generated in Parylene-coated glass devices adapting slanted T-junction geometry and dispersed in a continuous oil phase, the device could readily encapsulate and co-cultivate various subsets of a microbial community. A large number of droplets, up to ~1400 in a 10μmx5μm chamber, were generated within 3 seconds and packed very tightly in the chamber. A synthetic model system consisting of cross-feeding E. coli mutants was used to mimic various compositions of symbionts and other microbes in natural microbial communities. Our device was able to detect pair-wise symbiotic relationship when one partner accounted for as low as 1% of the total population or each symbiont was about 3% of the artificial community. In addition, murine fecal microbial samples were cultivated on the device and compositions of grown cultures were analyzed. Off-chip PCR of 16S rRNA genes with universal bacterial primers was carried out on retrieved co-cultures to determine species profiles of the cultivated subsets of the microbiota. It was shown that only a small number of species from the original sample were able to grow together in compartmentalized droplets under the tested conditions. This parallelized microfluidic compartmentalization and co-cultivation platform can also be extended for the investigation of microbial communities in other environments.