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Most experiments in screening and process development are performed in shaken bioreactors. Today, microtiter plates are the preferred vessels for small-scale microbial cultivations in high throughput, even though they have never been optimized for this purpose. To interpret the experimental results correctly and to obtain a base for a meaningful scale-up, sufficient oxygen supply to the culture liquid is crucial. For shaken bioreactors this problem can generally be addressed by the introduction of baffles. Therefore, the focus of this study is to investigate how baffling and the well geometry affect the maximum oxygen transfer capacity (OTR(max)) in microtiter plates. On a 48-well plate scale, 30 different cross-section geometries of a well were studied. It could be shown that the introduction of baffles into the common circular cylinder of a microtiter plate well doubles the maximum oxygen transfer capacity, resulting in values above 100 mmol/L/h (k(L)a > 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-monitoring systems such as the BioLector. This technology performs fiber-optical measurements through the well bottom, therefore requires a constant liquid height at all shaking frequencies. Ultimately, a six-petal flower-shaped well geometry was shown to be the optimal solution taking into account all aforementioned criteria. With its favorable culture conditions and the possibility for unrestricted online monitoring, this novel microtiter plate is an efficient tool to gain meaningful results for interpreting and scaling-up experiments in clone screening and bioprocess development.

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 strategy to cultivate both prokaryotic and eukaryotic cells. On the contrary, for small-scale cultivations, much simpler batch experiments with no process control are performed. This lack of process control often hinders researchers to scale-up and scale-down fermentation experiments, because the microbial metabolism and thereby the growth and production kinetics drastically changes depending on the cultivation strategy applied. While small-scale batches are typically performed highly parallel and in high throughput, large-scale cultivations demand sophisticated equipment for process control which is in most cases costly and difficult to handle. Currently, there is no technical system on the market that realizes simple process control in high throughput. The novel concept of a microfermentation system described in this work combines a fiber-optic online-monitoring device for microtiter plates (MTPs)--the BioLector technology--together with microfluidic control of cultivation processes in volumes below 1 mL. In the microfluidic chip, a micropump is integrated to realize distinct substrate flow rates during fed-batch cultivation in microscale. Hence, a cultivation system with several distinct advantages could be established: (1) high information output on a microscale; (2) many experiments can be performed in parallel and be automated using MTPs; (3) this system is user-friendly and can easily be transferred to a disposable single-use system. This article elucidates this new concept and illustrates applications in fermentations of Escherichia coli under pH-controlled and fed-batch conditions in shaken MTPs.

BackgroundThe efficiency of biotechnological production processes depends on selecting the best performing microbial strain and the optimal cultivation conditions. Thus, many experiments have to be conducted, which conflicts with the demand to speed up drug development processes. Consequently, there is a great need for high-throughput devices that allow rapid and reliable bioprocess development. This need 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. To further improve the application of MTPs as microbioreactors, in this paper, the BioLector technology is combined with microfluidic bioprocess control in MTPs. To realize a user-friendly system for routine laboratory work, disposable microfluidic MTPs are utilized which are actuated by a user-friendly pneumatic hardware.ResultsThis novel microfermentation system was tested in pH-controlled batch as well as in fed-batch fermentations of Escherichia coli. The pH-value in the culture broth could be kept in a narrow dead band of 0.03 around the pH-setpoint, by pneumatically dosing ammonia solution and phosphoric acid to each culture well. Furthermore, fed-batch cultivations with linear and exponential feeding of 500 g/L glucose solution were conducted. Finally, the scale-up potential of the microscale fermentations was evaluated by comparing the obtained results to that of fully controlled fermentations in a 2 L laboratory-scale fermenter (working volume of 1 L). The scale-up was realized by keeping the volumetric mass transfer coefficient kLa constant at a value of 460 1/h. The same growth behavior of the E. coli cultures could be observed on both scales.ConclusionIn microfluidic MTPs, pH-controlled batch as well as fed-batch fermentations were successfully performed. The liquid dosing as well as the biomass growth kinetics of the process-controlled fermentations agreed well both in the microscale and laboratory scale. In conclusion, a user-friendly and disposable microfluidic system could be established which allows scaleable, fully controlled and fully monitored fermentations in working volumes below 1 milliliter.

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