Microchemostat array with small-volume fraction replenishment for steady-state microbial culture

Abstract

A chemostat is a bioreactor in which microorganisms can be cultured at steady-state by controlling the rate of culture medium inflow and waste outflow, thus maintaining media composition over time. Even though many microbial studies could greatly benefit from studying microbes in steady-state conditions, high instrument cost, complexity, and large reagent consumption hamper the routine use of chemostats. Microfluidic-based chemostats (i.e. microchemostats) can operate with significantly smaller reagent consumption while providing accurate chemostatic conditions at orders of magnitude lower cost compared to conventional chemostats. Also, microchemostats have the potential to significantly increase the throughput by integrating arrays of microchemostats. We present a microchemostat array with a unique two-depth culture chamber design that enables small-volume fraction replenishment of culture medium as low as 1% per replenishment cycle in a 250 nl volume. A system having an array of 8 microchemostats on a 40 × 60 mm² footprint could be automatically operated in parallel by a single controller unit as a demonstration for potential high throughput microbial studies. The model organism, Saccharomyces cerevisiae, successfully reached a stable steady-state of different cell densities as a demonstration of the chemostatic functionality by programming the dilution rates. Chemostatic functionality of the system was further confirmed by quantifying the budding index as a function of dilution rate, a strong indicator of growth-dependent cell division. In addition, the small-volume fraction replenishment feature minimized the cell density fluctuation during the culture. The developed system provides a robust, low-cost, and higher throughput solution to furthering studies in microbial physiology.