Bioreactor Assembly for Continuous Culture of Complex Fecal Communities

The microbiota, especially bacteria, respond to various environmental exposures such as micro and macronutrients, pharmacological compounds, and inflammatory mediators, which dynamically alter community composition and microbial metabolic output. Understanding how physiological culture conditions affect microbial communities and diversity and their metabolic capacity will contribute important knowledge of their impact on health and diseases. Here, we present a protocol adapted from the template published by Auchtung et al. to create mini-bioreactor arrays that can cultivate complex fecal communities, define bacterial consortiums or single strains under precise conditions, including nutrient availability, temperature, flow rate, pH, and oxygen content. We describe the process for building the mini-bioreactor system, including adaptations to improve limitations in the published protocol. We also discuss the setup of the mini-bioreactor system under anaerobic conditions with the use of MEGA media (adapted from Han et al.), which is a rich media supporting the growth of diverse bacteria. We describe the inoculation of gut humanized mouse fecal samples into the mini-bioreactor system, followed by the establishment of complex community cultures within the mini-bioreactor system, which can be grown under continuous flow with aseptic sampling to monitor community composition. This system is adaptable to dietary changes and other cultural modifications. The techniques described here allow for the characterization of diverse, fastidious fecal communities under dynamic conditions or in response to perturbation in isolation from host-derived factors.