Microfluidic biolector—microfluidic bioprocess control in microtiter plates

Abstract

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.

Pitfalls in optical on-line monitoring for high-throughput screening of microbial systems

“…However, before the cells entered the stationary phase a second drop of the DOT signal is obtained for all three clones. This decrease, accompanied by an increase of the scattered light signal, is caused by the respiration of acetate accumulated during the cultivation [40]. The complete consumption of this by-product after 12.5, 14.5 and 15 h by E. coli mCherry, FbFP and YFP, respectively, leads to the final recovery of the DOT to values of 95–105% air saturation.…”

“…Performing cultivations in fed-batch mode avoids oxygen limitations, too. Controlled release systems [44], enzyme based fed-batch media [45], or microfluidic systems for MTPs [40] are convenient solutions. Nonetheless, this study does not aim for kinetic results so that an adjustment of the conditions was not necessary.…”

High-throughput fermentation screening for the yeast Yarrowia lipolytica with real-time monitoring of biomass and lipid production

“…However, the optimal growth conditions and genetic modifications for a laboratory setting are not necessarily the best-adapted for an industrial setting, and even slight deviations from the optimum can seriously affect industrial productivity. In particular, an erroneous assessment of growth and production conditions in the upstream development phase could greatly impair the economic viability of the bioprocess [19]. For this reason, high-throughput screening techniques and small-scale cultivation systems have been developed to accelerate process development and decrease costs by avoiding scale-up issues [20, 21].…”

“…It mimics the oxygen transfer rate and growth obtained in bioreactors but also enables the monitoring of fluorescence signals during fermentation. Fermentations conducted in the BioLector have led to protein production similar to that obtained in a 1.4 L bioreactor [19, 22]. Using the BioLector, it is possible to have the best of both worlds: like a bioreactor, it has the ability to track growth conditions in real time, and like standard microtiter plates, it enables rapid, high-throughput cultivation with online fluorescence monitoring.…”

Measuring growth rate in high-throughput growth phenotyping

“…This experimental setup will in particular provide valuable comparative data if combined with molecular reporters as in high content screening [51]. Furthermore, micro-fluidic systems [52] will most likely be used for gradually or distinctly altering media composition to study instantaneous growth rates during metabolic transitions. All these technical advances are important to meet the soon coming explosion of simulated data on growth rates in relation to genetic and environmental perturbations via the development of systems biology full-scale cellular models.…”