Microfluidic bioprocess control in baffled microtiter plates

  • Microfluidische Regelung von Bioprozessen in Mikrotiterplatten mit Schikanen

Funke, Matthias; Büchs, Jochen (Thesis advisor)

Aachen : Publikationsserver der RWTH Aachen University (2011)
Dissertation / PhD Thesis

Aachen, Techn. Hochsch., Diss., 2010


The efficiency of biotechnological production processes depends on selecting the best performing biocatalyst and the optimal operation conditions. Thus, during the screening and process development phases, many experiments have to be conducted, which conflicts with the demand to speed up drug development processes. This conflict is addressed, for example, by the fiber-optic online-monitoring system BioLector which utilizes the wells of shaken microtiter plates (MTPs) as small-scale fermenters. In this thesis, the BioLector technology is enhanced by incorporating microfluidic bioprocess control in design-optimized, baffled MTPs. Today, shaken MTPs are the preferred vessels for microbial cultivations in high throughput. Although, oxygen transfer in shaken bioreactors is often insufficient, this problem can generally be addressed by the introduction of baffles. Therefore, the focus of this study is to investigate how baffling affects the oxygen transfer in MTPs. On a 48-well plate scale, 30 different cross-section geometries of MTP wells were studied. It could be shown that the introduction of baffles into the circular cylinder of a MTP well doubles the maximum oxygen transfer capacity (OTRmax), resulting in values above 100 mmol/L/h (kLa > 600 1/h). To also guarantee a high volume for microbial cultivation, it is important to maximize the filling volume applicable during orbital shaking. Additionally, the liquid height at the well bottom was examined, which is a decisive parameter for online measurement by the BioLector. Ultimately, a six-petal flower-shape was identified as the optimal well geometry. Up to now, the geometry of baffles has neither been standardized, nor has their influence on hydrodynamics and cultivation conditions been systematically described. However, in this work a novel correlation was established by relating the measured values of OTRmax and filling height to the corresponding perimeter of the well cross-section area. This correlation systematically showed the influence of baffles in shaken vessels. Furthermore, it allows one to define the perimeter of the well cross-section area as the actual criterion for the so-called "degree of baffling". Additionally, a maximum baffling degree can be identified which should not be exceeded in order to avoid irregular liquid behavior and a decrease in the OTRmax. Consequently, this perimeter criterion provides a helpful tool to describe different baffle geometries and design new baffles. In industrial-scale biotechnological processes, the active control of the pH-value combined with the controlled feeding of substrate solutions (fed-batch) is the standard cultivation strategy. On the contrary, for small-scale cultivations, much simpler batch experiments with no process control are performed. This conflict between the scales often hinders researchers to scale up and scale down fermentation experiments. While small-scale batches are typically performed in high throughput, large-scale cultivations demand sophisticated equipment for process control. Currently, there is no technical system on the market that solves this conflict and realizes complete process control in high throughput. The novel concept of the microfermentation system described in this work combines the BioLector system together with microfluidic control of cultivation processes in volumes below 1 mL. To achieve this, in cooperation with the Institute for Materials in Electrical Engineering 1 of the RWTH Aachen University, a concept was developed in which microfluidic chips replace the bottom of conventional MTPs. The suitability of this system has been proven by pH-controlled and fed-batch fermentations of Escherichia coli in the microfluidic MTPs. Thereby, the aim was to establish a novel technology but simultaneously to focus on their use in routine laboratory work. Therefore, ready-to-use culture devices and user-friendly actuator hardware have been developed. Moreover, the scale-up potential of this system has been shown by obtaining equivalent fermentation results compared to a 1 L laboratory-scale stirred tank reactor. The developed microfermentation system combines four advantages: (1) improved microbioreactor design by applying the baffled MTPs, (2) microfluidic process control in disposable MTPs, (3) user-friendly system for connecting the microfluidic MTP to the pneumatic actuator hardware, (4) advanced online-monitoring by the BioLector technology. Integrating the aforementioned properties into one system allows microfluidic MTPs to be used as disposable, ready-to-use cultivation vessels in a user-friendly, "plug-and-cultivate" microfermentation system. This technology narrows the gap between microscale and production scale, since it allows scalable, fully controlled and fully monitored fermentations in working volumes below 1 milliliter. In conclusion, the developed system is a valuable tool for meaningful screening and process development.