04 Fakultät Energie-, Verfahrens- und Biotechnik
Permanent URI for this collectionhttps://elib.uni-stuttgart.de/handle/11682/5
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Item Open Access Optimizing mass transfer in multiphase fermentation : the role of drag models and physical conditions(2023) Mast, Yannic; Wild, Moritz; Takors, RalfDetailed knowledge of the flow characteristics, bubble movement, and mass transfer is a prerequisite for the proper design of multiphase bioreactors. Often, mechanistic spatiotemporal models and computational fluid dynamics, which intrinsically require computationally demanding analysis of local interfacial forces, are applied. Typically, such approaches use volumetric mass-transfer coefficient (kLa) models, which have demonstrated their predictive power in water systems. However, are the related results transferrable to multiphase fermentations with different physicochemical properties? This is crucial for the proper design of biotechnological processes. Accordingly, this study investigated a given set of mass transfer data to characterize the fermentation conditions. To prevent time-consuming simulations, computational efforts were reduced using a force balance stationary 0-dimension model. Therefore, a competing set of drag models covering different mechanistic assumptions could be evaluated. The simplified approach of disregarding fluid movement provided reliable results and outlined the need to identify the liquid diffusion coefficients in fermentation media. To predict the rising bubble velocities uB, the models considering the Morton number (Mo) showed superiority. The mass transfer coefficient kL was best described using the well-known Higbie approach. Taken together, the gas hold-up, specific surface area, and integral mass transfer could be accurately predicted.Item Open Access Getting the right clones in an automated manner : an alternative to sophisticated colony-picking robotics(2024) Hägele, Lorena; Pfleger, Brian F.; Takors, RalfIn recent years, the design-build-test-learn (DBTL) cycle has become a key concept in strain engineering. Modern biofoundries enable automated DBTL cycling using robotic devices. However, both highly automated facilities and semi-automated facilities encounter bottlenecks in clone selection and screening. While fully automated biofoundries can take advantage of expensive commercially available colony pickers, semi-automated facilities have to fall back on affordable alternatives. Therefore, our clone selection method is particularly well-suited for academic settings, requiring only the basic infrastructure of a biofoundry. The automated liquid clone selection (ALCS) method represents a straightforward approach for clone selection. Similar to sophisticated colony-picking robots, the ALCS approach aims to achieve high selectivity. Investigating the time analogue of five generations, the model-based set-up reached a selectivity of 98 ± 0.2% for correctly transformed cells. Moreover, the method is robust to variations in cell numbers at the start of ALCS. Beside Escherichia coli , promising chassis organisms, such as Pseudomonas putida and Corynebacterium glutamicum , were successfully applied. In all cases, ALCS enables the immediate use of the selected strains in follow-up applications. In essence, our ALCS approach provides a ‘low-tech’ method to be implemented in biofoundry settings without requiring additional devices.Item Open Access Predicting by-product gradients of baker’s yeast production at industrial scale : a practical simulation approach(2020) Sarkizi Shams Hajian, Christopher; Haringa, Cees; Noorman, Henk; Takors, RalfScaling up bioprocesses is one of the most crucial steps in the commercialization of bioproducts. While it is known that concentration and shear rate gradients occur at larger scales, it is often too risky, if feasible at all, to conduct validation experiments at such scales. Using computational fluid dynamics equipped with mechanistic biochemical engineering knowledge of the process, it is possible to simulate such gradients. In this work, concentration profiles for the by-products of baker’s yeast production are investigated. By applying a mechanistic black-box model, concentration heterogeneities for oxygen, glucose, ethanol, and carbon dioxide are evaluated. The results suggest that, although at low concentrations, ethanol is consumed in more than 90% of the tank volume, which prevents cell starvation, even when glucose is virtually depleted. Moreover, long exposure to high dissolved carbon dioxide levels is predicted. Two biomass concentrations, i.e., 10 and 25 g/L, are considered where, in the former, ethanol production is solely because of overflow metabolism while, in the latter, 10% of the ethanol formation is due to dissolved oxygen limitation. This method facilitates the prediction of the living conditions of the microorganism and its utilization to address the limitations via change of strain or bioreactor design or operation conditions. The outcome can also be of value to design a representative scale-down reactor to facilitate strain studies.Item Open Access Investigation of tracer gas transport in a new numerical model of lung acini(2022) Schmidt, Christoph; Joppek, Christoph; Trinkmann, Frederik; Takors, Ralf; Cattaneo, Giorgio; Port, JohannesObstructive pulmonary diseases are associated with considerable morbidity. For an early diagnosis of these diseases, inert gas washouts can potentially be used. However, the complex interaction between lung anatomy and gas transport mechanisms complicates data analysis. In order to investigate this interaction, a numerical model, based on the finite difference method, consisting of two lung units connected in parallel, was developed to simulate the tracer gas transport within the human acinus. Firstly, the geometries of the units were varied and the diffusion coefficients ( D ) were kept constant. Secondly, D was changed and the geometry was kept constant. Furthermore, simple monoexponential growth functions were applied to evaluate the simulated data. In 109 of the 112 analyzed curves, monoexponential function matched simulated data with an accuracy of over 90%, potentially representing a suitable numerical tool to predict transport processes in further model extensions. For total flows greater than 5 × 10 -4 ml/s, the exponential growth constants increased linearly with linear increasing flow to an accuracy of over 95%. The slopes of these linear trend lines of 1.23 µl -1 ( D = 0.6 cm 2 /s), 1.69 µl -1 ( D = 0.3 cm 2 /s), and 2.25 µl -1 ( D = 0.1 cm 2 /s) indicated that gases with low D are more sensitive to changes in flows than gases with high D .Item Open Access Simulated oxygen and glucose gradients as a prerequisite for predicting industrial scale performance a priori(2020) Kuschel, Maike; Takors, RalfTransferring bioprocesses from lab to industrial scale without loss of performance is key for the successful implementation of novel production approaches. Because mixing and mass transfer is usually hampered in large scale, cells experience heterogeneities eventually causing deteriorated yields, that is, reduced titers, productivities, and sugar‐to‐product conversions. Accordingly, reliable and easy‐to‐implement tools for a priori prediction of large‐scale performance based on dry and wet‐lab tests are heavily needed. This study makes use of computational fluid dynamic simulations of a multiphase multi‐impeller stirred tank in pilot scale. So‐called lifelines, records of 120,000 Corynebacterium glutamicum cells experiencing fluctuating environmental conditions, were identified and used to properly design wet‐lab scale‐down (SD) devices. Physical parameters such as power input, gas hold up, kLa, and mixing time showed good agreement with experimental measurements. Analyzing the late fed‐batch cultivation revealed that the complex double gradient of glucose and oxygen can be translated into a wet‐lab SD setup with only few compartments. Most remarkably, the comparison of different mesh sizes outlined that even the coarsest approach with a mesh density of 1.12×105#/m3 was sufficient to properly predict physical and biological readouts. Accordingly, the approach offers the potential for the thorough analysis of realistic industrial case scenarios.Item Open Access Streamlining the analysis of dynamic 13C-labeling patterns for the metabolic engineering of corynebacterium glutamicum as L-histidine production host(2020) Feith, André; Schwentner, Andreas; Teleki, Attila; Favilli, Lorenzo; Blombach, Bastian; Takors, RalfToday’s possibilities of genome editing easily create plentitudes of strain mutants that need to be experimentally qualified for configuring the next steps of strain engineering. The application of design-build-test-learn cycles requires the identification of distinct metabolic engineering targets as design inputs for subsequent optimization rounds. Here, we present the pool influx kinetics (PIK) approach that identifies promising metabolic engineering targets by pairwise comparison of up- and downstream 13C labeling dynamics with respect to a metabolite of interest. Showcasing the complex l-histidine production with engineered Corynebacterium glutamicum l-histidine-on-glucose yields could be improved to 8.6 ± 0.1 mol% by PIK analysis, starting from a base strain. Amplification of purA, purB, purH, and formyl recycling was identified as key targets only analyzing the signal transduction kinetics mirrored in the PIK values.Item Open Access Synthetic co-culture in an interconnected two-compartment bioreactor system : violacein production with recombinant E. coli strains(2024) Müller, Tobias; Schick, Simon; Klemp, Jan-Simon; Sprenger, Georg A.; Takors, RalfThe concept of modular synthetic co-cultures holds considerable potential for biomanufacturing, primarily to reduce the metabolic burden of individual strains by sharing tasks among consortium members. However, current consortia often show unilateral relationships solely, without stabilizing feedback control mechanisms, and are grown in a shared cultivation setting. Such ‘one pot’ approaches hardly install optimum growth and production conditions for the individual partners. Hence, novel mutualistic, self-coordinating consortia are needed that are cultured under optimal growth and production conditions for each member. The heterologous production of the antibiotic violacein (VIO) in the mutually interacting E. coli - E. coli consortium serves as an example of this new principle. Interdependencies for growth control were implemented via auxotrophies for L-tryptophan and anthranilate (ANT) that were satisfied by the respective partner. Furthermore, VIO production was installed in the ANT auxotrophic strain. VIO production, however, requires low temperatures of 20-30 °C which conflicts with the optimum growth temperature of E. coli at 37 °C. Consequently, a two-compartment, two-temperature level setup was used, retaining the mutual interaction of the cells via the filter membrane-based exchange of medium. This configuration also provided the flexibility to perform individualized batch and fed-batch strategies for each co-culture member. We achieved maximum biomass-specific productivities of around 6 mg (g h) -1 at 25 °C which holds great promise for future applications.Item Open Access Growth-rate dependency of ribosome abundance and translation elongation rate in Corynebacterium glutamicum differs from that in Escherichia coli(2023) Matamouros, Susana; Gensch, Thomas; Cerff, Martin; Sachs, Christian C.; Abdollahzadeh, Iman; Hendriks, Johnny; Horst, Lucas; Tenhaef, Niklas; Tenhaef, Julia; Noack, Stephan; Graf, Michaela; Takors, Ralf; Nöh, Katharina; Bott, MichaelBacterial growth rate (µ) depends on the protein synthesis capacity of the cell and thus on the number of active ribosomes and their translation elongation rate. The relationship between these fundamental growth parameters have only been described for few bacterial species, in particular Escherichia coli . Here, we analyse the growth-rate dependency of ribosome abundance and translation elongation rate for Corynebacterium glutamicum , a gram-positive model species differing from E. coli by a lower growth temperature optimum and a lower maximal growth rate. We show that, unlike in E. coli , there is little change in ribosome abundance for µ <0.4 h -1 in C. glutamicum and the fraction of active ribosomes is kept above 70% while the translation elongation rate declines 5-fold. Mathematical modelling indicates that the decrease in the translation elongation rate can be explained by a depletion of translation precursors.