Microfluidic devices for bioprocess control in microtiter plates

Biotechnological processes are a key technology in the synthesis of high-grade chemicals and pharmaceuticals. The efficiency of production processes is determined during the development phase by the selection of biocatalysts, either enzymes or microorganisms, and the process conditions. But the development of biocatalytical processes is often time-consuming. The reason is the necessity to first identify a suitable biocatalyst and then to find the process parameters for an efficient expression of the biocatalyst. Since the number of screening experiments may amount to several thousand, microtiter plates (MTP) and related measurement technologies have recently attracted a lot of interest. Thus, methods for online monitoring of important process parameters are available. However, the established methods either lack process control and thus comparability to industrial scale fermenters on the one hand or sufficiently high throughput on the other hand. Automated fermentations in the submilliliter scale with an integrated online monitoring and fluid supply are regarded as a promising approach to overcome this restriction in process development. In this work, microfluidic devices for MTP based microbioreactors have been developed, fabricated and characterized. The devices are designed for the use in conjunction with established optical online monitoring systems and, in addition, four sensor electrodes can be integrated. The microfluidic devices have been realized in polydimethylsiloxane (PDMS) by soft lithographic processes and in the negative tone photoresist SU 8 using multilayer photolithographic processes. The dispensing characteristics have been analyzed in numeric simulations and evaluated in fluorescence based measurements. The microbioreactors have been equipped with an interface to external actuation and measurement systems, in order to control cultivation processes in up to 24 wells in parallel. The microfluidic devices dispense aqueous pH control media ranging from less than 5 nl to more than 183 nl using dispensing times of 18 ms to 100 ms. Integrated micropumps are able to supply nutrient solutions in volumes of approximately 150 nl with a standard deviation of less than 5%. The reproducible dispensing of fluids with viscosities of up to 109 mPa s has been shown. The microbioreactors have been successfully applied to pH controlled fermentations of E. coli in TB medium. By dispensing sodium hydroxide and phosphoric acid, the pH in controlled cultures could be maintained between 6.85 and 7.03 with a set point of 7.0. In contrast, the pH in uncontrolled cultures varied between 6.46 and 8.83. Thus, the microbioreactor has proven its capability of controlling the pH within narrow tolerances. For the first time, the described devices allow pH controlled and fed-batch fermentations in a MTP based microbioreactor with an integrated microfluidic control. The microbioreactor reaches a degree of parallelization, which is comparable to standard microtiter plates. Moreover, in conjunction with optical online monitoring, the precise and flexible fluid supply provides an experimental platform, which allows evaluating different fermentation strategies