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Microbial screening experiments are of utmost importance for developing biotechnological processes. The cultivation parameters for selecting the most suited microorganisms during these screening experiments should match the parameters for the subsequent production process as exact as possible. It is absolutely necessary, to apply comparable cultivation conditions in small-scale screenings and large-scale production processes, to ensure a meaningful analysis of the screening experiments as well as a successful scale-up of its results. In the presented work, different facets of screening processes were analyzed and solutions for their optimization regarding the aforementioned general principles were investigated. One key factor to improve the comparability of screening and production-scale experiments is to establish an online monitoring of cultivation parameters not only in large- but also in small-scale cultivations. To enhance the online information obtained during screening and process development in shake flasks, the RAMOS device for measuring respiration activities in shake flaks and a fiber optical, online pH-measurement technique were successfully combined. To further improve the comparability between the different scales and to enable a more reliable scale-up of experiments, the cultivation strategy (i.e. the progression of pH-value and substrate concentration during the cultivation) has to be comparable in small- and in large-scales. Thus, the in large-scale applied pH-control and fed-batch operational mode have to be adapted to small scale screening experiments. A disc-shaped polymer-based controlled-release system for pH-control in shake flasks was developed and applied in this thesis. It consists of a polymer matrix in which sodium carbonate as pH-control reagent is encased. When applied in cultivation media, this system releases sodium carbonate in pre-defined kinetics. With this system, it was possible to substantially reduce the buffer concentrations in shake flask cultivations of Escherichia coli, while the pH-values remained in the physiological range of microbial growth. An additional physiological effect of the pH-value is its influence on the growth behavior of the microorganisms and thereby especially on the duration of the lag-phase. Different lag times of the microorganisms considerably affect the outcome of screening processes. In this work it could be shown that the initial pH-value of the cultivation media has an enormous strain dependent effect on the lag time of E. coli cultures. For three E. coli strains a lower initial pH-value resulted in a shorter lag phase and one strain showed the opposite behavior. This parameter should be considered in the design of production processes as well as of screening experiments. Another analyzed facet of the screening process was the microbial growth in precultures. It could be demonstrated, that differences in the inoculum from precultures in shaken bioreactors have a tremendous effect on the microbial growth and thus on rational design of screening processes. Therefore, a new technique applying fed-batch mode in high-throughput precultivations for equalizing the initial parameters of subsequent screening experiments was introduced. For fed-batch cultivation in shake flasks, glucose containing polymer-based controlled-release discs were applied. For high-throughput applications a new fed-batch microtiter plate, with immobilized polymer-based controlled-release systems at the bottom of each well were presented. The newly developed fed-batch precultivation method enables equalized growth of all screened strains and will generate, therefore, more relevant and reliable data in subsequent main screening experiments. The feasibility of the presented concept has been proven for cultivations of E. coli and Hansenula polymorpha. All these results demonstrate the importance of choosing the correct cultivation parameters for a successful microbial screening. Especially the control of the pH-value and the controlled-release of substrate are important for several aspects of screening experiments. The systems and methodologies described in the current work significantly improve screening procedures and the meaningful analysis and scale-up of the obtained results. |
