A microbioreactor with a volume of microliters is fabricated out of poly(dimethylsiloxane) (PDMS) and glass. Aeration of microbial cultures is through a gas-permeable PDMS membrane. Sensors are integrated for on-line measurement of optical density (OD), dissolved oxygen (DO), and pH. All three parameter measurements are based on optical methods. Optical density is monitored via transmittance measurements through the well of the microbioreactor while dissolved oxygen and pH are measured using fluorescence lifetime-based sensors incorporated into the body of the microbioreactor. Bacterial fermentations carried out in the microbioreactor under well-defined conditions are compared to results obtained in a 500-mL bench-scale bioreactor. It is shown that the behavior of the bacteria in the microbioreactor is similar to that in the larger bioreactor. This similarity includes growth kinetics, dissolved oxygen profile within the vessel over time, pH profile over time, final number of cells, and cell morphology. Results from off-line analysis of the medium to examine organic acid production and substrate utilization are presented. By changing the gaseous environmental conditions, it is demonstrated that oxygen levels within the microbioreactor can be manipulated. Furthermore, it is demonstrated that the sensitivity and reproducibility of the microbioreactor system are such that statistically significant differences in the time evolution of the OD, DO, and pH can be used to distinguish between different physiological states. Finally, modeling of the transient oxygen transfer within the microbioreactor based on observed and predicted growth kinetics is used to quantitatively characterize oxygen depletion in the system.

A multiplexed microbioreactor system for parallel operation of multiple microbial fermentation is described. The system includes miniature motors for magnetic stirring of the microbioreactors and optics to monitor the fermentation parameters optical density (OD), dissolved oxygen (DO), and pH, in-situ and in real time. The microbioreactors are fabricated out of poly(methylmethacrylate)(PMMA) and poly(dimethylsiloxane)(PDMS), and have a working volume of 150 microl. Oxygenation of the cells occurs through a thin PDMS membrane at the top of the reactor chamber. Stirring is achieved with a magnetic spin bar in the reactor chamber. Parallel microbial fermentations with Escherichia coli are carried out in four stirred microbioreactors and demonstrate the reproducible performance of the multiplexed system. The profiles for OD, DO, and pH compare favourably to fermentations performed in bioreactor systems with multiple bench-scale reactors. Finally, the multiplexed system is used to compare two different reactor designs, demonstrating that the reproducibility of the system permits the quantification of microbioreactor performance.

We have developed an integrated array of microbioreactors, with 100 microL working volume, comprising a peristaltic oxygenating mixer and microfluidic injectors. These integrated devices were fabricated in a single chip and can provide a high oxygen transfer rate (k(L)a approximately 0.1 s(-1)) without introducing bubbles, and closed loop control over dissolved oxygen and pH (+/-0.1). The system was capable of supporting eight simultaneous Escherichia coli fermentations to cell densities greater than 13 g-dcw L(-1) (1 cm OD(650 nm) > 40). This cell density was comparable to that achieved in a 4 litre reference fermentation, conducted with the same strain, in a bench scale stirred tank bioreactor and is more than four times higher than cell densities previously achieved in microbioreactors. Bubble free oxygenation permitted near real time optical density measurements which could be used to observe subtle changes in the growth rate and infer changes in the state of microbial genetic networks. Our system provides a platform for the study of the interaction of microbial populations with different environmental conditions, which has applications in basic science and industrial bioprocess development. We leverage the advantages of microfluidic integration to deliver a disposable, parallel bioreactor in a single chip, rather than robotically multiplexing independent bioreactors, which opens a new avenue for scaling small scale bioreactor arrays with the capabilities of bench scale stirred tank reactors.

This work reports on an instrument capable of supporting automated microscale continuous culture experiments. The instrument consists of a plastic-PDMS device capable of continuous flow without volume drift or evaporation. We apply direct computer controlled machining and chemical bonding fabrication for production of fluidic devices with a 1 mL working volume, high oxygen transfer rate (k(L)a≈0.025 s(-1)), fast mixing (2 s), accurate flow control (±18 nL), and closed loop control over temperature, cell density, dissolved oxygen, and pH. Integrated peristaltic pumps and valves provide control over input concentrations and allow the system to perform different types of cell culture on a single device, such as batch, chemostat, and turbidostat continuous cultures. Continuous cultures are demonstrated without contamination for 3 weeks in a single device and both steady state and dynamically controlled conditions are possible.

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