Matthias Funke, Jochen Büchs, Andreas Buchenauer, Wilfried Mokwa, Frank Kensy, Carsten Müller:
Microfluidic Controlled Fermentations in Microtiter Plates
14th European Congress on Biotechnology, Barcelona, Spain, 16.09.2009
In clone screening and even more in bioprocess development it is mandatory to mimic large-scale production processes to obtain meaningful results for scale-up from small scale experiments. Since high throughput, easy handling and low costs are aspired in these applications, microtiter plates are nowadays the predominant vessel for screening and high-throughput experiments. However, one important disadvantage of biotechnological cultivations in microtiter plates is the discrepancy in process control compared to industrial production scale. Whereas many large scale fermentations use pH-control and/or substrate feeding, microtiter plates are still limited to uncontrolled batch fermentations. In screening applications the selection of unfavourable strains or culture conditions might be the result, since batch results are in many cases not transferable to industrial fed-batch processes. In process development this discrepancy leads to more expensive and time-consuming experiments in laboratory-scale stirred tank reactors. Here, a new technique is presented to perform controlled cultivations in the small scale of a microtiter plate. The combination of a microfluidic device and a 48-well microtiter plate establishes the possibility to dose volumes down to nL-scale to each well individually. A microfluidic chip substitutes the bottom of a conventional microtiter plate. It incorporates channels in dimensions down to 100 nm as well as independently triggered valves and pumps. The microtiter plate with microfluidic control can easily be connected with the actuator hardware by simply clamping the plate on a shaker tray with pneumatic connections. Thus a user-friendly handling of the plate can be guaranteed. With this system, the dosage of acid and base as well as the feeding of high concentrated substrate solutions to each well of the microtiter plate individually are possible. Fermentations with pH control as well as fed-batch fermentations with different feeding strategies (constant, linear increasing, exponential) have been performed with Escherichia coli. The results obtained with the microfluidic controlled microtiter plates can be reproduced in parallel fermentations in 1L laboratory-scale stirred tank reactor. Besides the microfluidic control, the fermentations are monitored online with the BioLector-technology. This fiber-optic measurement system, developed in our group, allows an online monitoring of biomass (via scattered light detection), metabolism (e.g. via NADH fluorescence) and production (via GFP, YFP fluorescence). The combination of the microfluidics in microtiter plates and the BioLector online-monitoring technology allows high-throughput as well as data acquisition and process control in small scale comparable to industrial production processes.
microtiter plate; fermentation; pH-control; fed-batch; screening; process development