Bacterial growth and adaptation in microdroplet chemostats.

We describe herein microfluidic technology for manipulating and monitoring continuous growth of populations of bacteria. A system consisting of approximately ten input and output channels controls more than 100 microdroplet chemostats and enables the manipulation of chemical factors in each microchemostat independently over time. Herein, we characterize the dynamics of bacterial populations in microdroplet chemostats and cellular responses to a range of stable or changing antibiotic concentrations. This method allows for parallel, long-term studies of microbial ecology, physiology, evolution, and adaptation to chemical environments. The introduction of the chemostat by Leo Szilard was a milestone in the field of microbiology. Chemostats facilitate the continuous culture of bacteria, yeast, and algae by continuously replenishing a constant volume of fluid to maintain specific concentrations of cells and growth factors. Chemostats have facilitated a wide-range of studies, including microbial ecology, predator–prey dynamics, and the evolution of drug resistance. The consumption of large quantities of reagents and the significant operational challenges of traditional chemostats limit their use. Single-phase, microfluidic versions of chemostats minimize incubation volumes, and yet are limited by their complexity: the proportionality between the number of input/ output controls and the number of chemostats hamper large scale parallelization. Single-phase microfluidic systems are prone to biofilm formation, which makes them either singleuse devices or requiring additional steps to minimize cell adhesion. Droplet microfluidics offer a unique solution to creating many parallel chemostats. The earliest example of this technology in microbiology was first demonstrated by Joshua Lederberg nearly 60 years ago. In the interim, the field of microfluidics solved many of the technical challenges associated with using this approach to study microbes. Compartmentalizing cells and nutrients in microdroplets of liquid can reduce the complexity and cost of operating many parallel chemostats. Recently, bacteria have been incubated in droplets in channels over short time intervals, however sustained cell growth over hundreds of generations in a series of fully addressable microdroplets has not been possible. Herein, we describe an automated microdroplet system that transcends existing challenges and enables users to manipulate the chemical composition of droplets for longterm bacterial studies. The microfluidic system (Figure 1) performs three functions: 1) formation of microdroplets containing cells, reagents, and soluble growth factors; 2) cycling microdroplets for cell incubation and monitoring; and 3) splitting and fusing microdroplets to control the concentration of chemical factors over time. After loading the reservoirs with liquid samples, we used a source of pressure and external valves to regulate the flow of