Optimization of mutagenicity detection and inulosucrase production by monitoring the oxygen transfer rate in small-scale culture systems

The first part of this thesis focuses on the development and validation of the Ames RAMOS test, an in vitro test that detects mutagenic substances through monitoring of the oxygen transfer rate (OTR) of Salmonella test strains. The original Ames test is conducted on agar plates and evaluated through colony counting. To establish a direct connection between the two test formats, the area-specific oxygen transfer rate was monitored throughout an original Ames test on agar plates. The qualitative trend agreed well with the OTR during the Ames RAMOS test. When the Ames RAMOS test was further miniaturized to a 96-well format next, the relative error increased. The primary parameter responsible was identified as the inoculum cell count exposed to a test chemical. Literature data confirmed this phenomenon for other Ames test formats. However, increasing the inoculum cell count to reduce the statistical error reduced the separation efficiency of the Ames RAMOS test. The test protocol was thus optimized based on the lower limit of still detectable concentrations of a test substance. The resulting 96-well Ames RAMOS test was validated with 18 known chemicals, revealing a test sensitivity of 78.6 %. In addition to mutagenicity, the oxygen transfer rate allowed for the integrated cytotoxicity detection. As cytotoxic effects of test chemicals are a common cause of false negative results in the Ames test, the Ames RAMOS test offers a more efficient and resource-saving protocol than established protocols and particularly precise detection of weakly mutagenic chemicals. In the second part of this thesis, a process for producing the inulosucrase InuGB-V3 with Vibrio natriegens Vmax was developed. V. natriegens is known for its very high growth rate, enabling faster and, therefore, more economical processes. Initially, the influence of buffering and sodium chloride concentration on the growth rate of V. natriegens was investigated. Due to the high acetate formation observed in the presence of excess glucose, strong buffering was necessary to mitigate the risk of a pH inhibition. In order to avoid excess glucose conditions, a fed-batch process was aimed at for further process development. A glucose soft sensor based on the oxygen transfer rate was developed and validated to determine the feeding rate even at very low sample volumes. The expression of the inulosucrase InuGB-V3 was then analyzed as a function of the inducer concentration. This revealed significant differences in batch mode, which were not observed in fed-batch. A direct comparison of V. natriegens with the established expression host Escherichia coli was subsequently carried out in batch mode. Induction profiles were obtained for both organisms, whereby 25 % higher titers were achieved in V. natriegens. Overall, the second part of this work demonstrated both the suitability of V. natriegens as an expression host for InuGB-V3, and the efficiency and versatility of process development in small-scale

Optimizing recombinant protein expression via automated induction profiling in microtiter plates at different temperatures

Abstract Background Escherichia coli (E. coli) is the most abundant expression host for recombinant proteins. The production efficiency is dependent on a multitude of parameters. Therefore, high-throughput applications have become an increasingly frequent technique to investigate the main factors. Within this study, the effects of temperature, induction time and inducer concentration on the metabolic state and the product formation were extensively examined. Induction profiling of E. coli Tuner(DE3) pRhotHi-2-EcFbFP was performed in 48-well Flowerplates and standard 96-well plates using a robotic platform. In parallel shake flask cultivations, the respiration activity of the microorganisms was analyzed. Therefore, two online-monitoring systems were applied: the BioLector for microtiter plates and the RAMOS-device for shake flasks. The impact of different induction conditions on biomass and product formation as well as on the oxygen transfer rate was surveyed. Results Different optimal induction conditions were obtained for temperatures of 28, 30, 34, and 37 °C. The best inducer concentrations were determined to be between 0.05 and 0.1 mM IPTG for all investigated temperatures. This is 10–20 times lower than conventional guidelines suggest. The induction time was less relevant when the correct inducer concentration was chosen. Furthermore, there was a stronger impact on growth and respiration activity at higher temperatures. This indicated a higher metabolic burden. Therefore, lower IPTG concentrations were advantageous at elevated temperatures. Very similar results were obtained in standard 96-well plates. Conclusion Two online-monitoring systems were successfully used to investigate the optimal induction conditions for the E. coli Tuner(DE3) pRhotHi-2-EcFbFP strain (lacY deletion mutant) at four different temperatures. The experimental effort was reduced to a minimum by integrating a liquid handling robot. To reach the maximum product formation, a detailed induction analysis was necessary. Whenever the cultivation temperature was changed, the induction conditions have to be adapted. Due to the experimental options provided by the BioLector technology, it was found that the higher the cultivation temperature, the lower the inducer concentration that has to be applied

Phosphate limitation enhances malic acid production on nitrogen-rich molasses with Ustilago trichophora

Abstract Background An important step in replacing petrochemical products with sustainable, cost-effective alternatives is the use of feedstocks other than, e.g., pure glucose in the fermentative production of platform chemicals. Ustilaginaceae offer the advantages of a wide substrate spectrum and naturally produce a versatile range of value-added compounds under nitrogen limitation. A promising candidate is the dicarboxylic acid malic acid, which may be applied as an acidulant in the food industry, a chelating agent in pharmaceuticals, or in biobased polymer production. However, fermentable residue streams from the food and agricultural industry with high nitrogen content, e.g., sugar beet molasses, are unsuited for processes with Ustilaginaceae, as they result in low product yields due to high biomass and low product formation. Results This study uncovers challenges in evaluating complex feedstock applicability for microbial production processes, highlighting the role of secondary substrate limitations, internal storage molecules, and incomplete assimilation of these substrates. A microliter-scale screening method with online monitoring of microbial respiration was developed using malic acid production with Ustilago trichophora on molasses as an application example. Investigation into nitrogen, phosphate, sulphate, and magnesium limitations on a defined minimal medium demonstrated successful malic acid production under nitrogen and phosphate limitation. Furthermore, a reduction of nitrogen and phosphate in the elemental composition of U. trichophora was revealed under the respective secondary substrate limitation. These adaptive changes in combination with the intricate metabolic response hinder mathematical prediction of product formation and make the presented screening methodology for complex feedstocks imperative. In the next step, the screening was transferred to a molasses-based complex medium. It was determined that the organism assimilated only 25% and 50% of the elemental nitrogen and phosphorus present in molasses, respectively. Due to the overall low content of bioavailable phosphorus in molasses, the replacement of the state-of-the-art nitrogen limitation was shown to increase malic acid production by 65%. Conclusion The identification of phosphate as a superior secondary substrate limitation for enhanced malic acid production opens up new opportunities for the effective utilization of molasses as a more sustainable and cost-effective substrate than, e.g., pure glucose for biobased platform chemical production