(101f) Development of Continuous Culture Microbioreactors

In industrial fermentation processes, starting up a new production is usually preceded by a tremendous research effort in which for example the productivity of different candidate production strains is compared (=screening). Ranking of strains according to productivity resulting from the screening phase is crucial in selecting strains for full-scale production. Initially, screening is done in microtiter plates or by using shake flask cultures. Experiments are rather easy to set up, but only batch experiments are possible. The information gained per experiment is limited, and typically only end-point measurements are performed. The ranking of strains resulting from these experiments does sometimes not reflect results at industrial scale, for example because these batch type processes are characterized by more extreme forms of nutrient excess/limitation compared to the fed-batch processes used at larger scales. In a later stage of production process development, experiments are performed in bench scale reactors to investigate the influence of process conditions on productivity. Bench scale reactors (typically with a volume of 1 to 10 L) have the advantage that they allow on-line measurements, and they are flexible since they can be operated in batch or fed-batch, but also as a continuous culture. However, the effort needed to prepare, operate and subsequently clean bench scale reactors is vast.

Microbioreactors offer the possibility to circumvent many of the above-mentioned problems: (1) Scaling out microbioreactors to systems with many parallel reactors allows for high-throughput screening; (2) The working volumes are very small (μL to mL range), keeping costs for culture media low; (3) On-line measurements are possible for the most important culture variables (optical density (OD), dissolved oxygen (DO), pH); (4) The reactors can be fabricated from polymers, thus making them disposable after use which greatly reduces assembly, cleaning and sterilisation efforts, thereby further reducing cost; (5) Finally, for batch type microbioreactors, the fermentation variables compare favorably with bench scale reactors, which indicates that the right culture physiology can be maintained at small vs. larger scale. Therefore, the ranking of any strains tested can be preserved across scales, making the technology commercially viable.

This work reports on the development of a continuous culture microbioreactor platform that can perform experiments with yeast (Saccharomyces cerevisiae). Compared to a batch experiment, the continuous culture has the advantage that steady-state conditions can be achieved. Additionally, it should be possible to induce step changes in the dilution rate, forcing the culture from one steady-state to the other with continuous measurement of the important culture variables, thus leading to dynamic information on the behavior of the culture under well-controlled experimental conditions.

The microbioreactor ? with a volume of 100 μL ? is fabricated out of the polymers poly(methylmethacrylate) (PMMA) and poly(dimethylsiloxane) (PDMS). Dissolved oxygen (DO) and pH are both measured with fluorescent sensor spots. Optical density (OD) is measured in the reactor itself. Contrary to conventional bench-scale reactors, microbioreactors are designed to work bubble-free: aeration is done through a semi-permeable PDMS membrane. A simpler membrane fabrication methodology was developed, allowing for cheaper manufacturing. Proper mixing is essential for good cultivation results in the microbioreactors, since substrate gradients might lead to a varying (location dependent) metabolic state of the culture. In the projected volume range, convective mixing is difficult to achieve due to the small Reynolds numbers. On the other hand, the volume is too large to be able to rely on diffusion alone. Different methods for mixing the reactor contents were investigated. Finally, results of S. cerevisiae cultivations performed in the microbioreactor will be reported and compared to literature data.