Dissertationen/Habilitationen

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jlupub.ub.uni-giessen.de/handle/jlupub/64

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Concept study to develop a platform process for the production of antimicrobial peptides/proteins
(2023) Lappöhn, Carolin Anna
The increasing prevalence of antibiotic-resistant pathogens has created a demand for alternative treatments. However, the production of antimicrobial peptides and proteins (AMPs) in sufficient quantities for therapeutic use remains a significant bottleneck. This study introduces an innovative platform technology for the heterologous production, purification, and detection of AMPs. The technology is based on a small fusion tag, called the C-tag, composed of four amino acids (EPEA) fused to the C terminus of AMPs. Notably, using a complex model AMP, we demonstrated for the first time that the C-tag enhances the expression of this AMP in Escherichia coli compared to the unmodified peptide. Further process intensification increased the yield by nearly 30-fold compared to previous attempts with the same AMP. The primary advantage of the tag is apparent during product purification, leading to > 85% recovery and ~80% purity in a single step without compromising product activity, thus eliminating the need to remove the tag in a cost-intensive additional process step. In the subsequent parts of this study, we introduce, for the first time, an analytical immunoaffinity chromatography based on the C-tag. A detailed exploration of the critical process parameters using statistical optimization techniques enabled us to achieve 98.8 ± 0.1% analyte recovery. Validation of the method indicated high specificity, linearity, accuracy, and precision, underscored by a short sample analysis time, enabling high-throughput sample screening. Further characterization of the method revealed its limited ability to detect AMPs in acidic samples (pH < 2). Pretreatment strategies to overcome these limitations are discussed. Consequently, this study introduces a quantitative C-tag platform for the in-process monitoring of AMPs.
Einfluss der Oberflächenrauigkeit auf die mechanischen Eigenschaften hochorientierter kurzglasfaserverstärkter thermoplastischer Polymere
(2023) van Roo, Tamara
Das mechanische Verhalten spritzgegossener, kurzglasfaserverstärkter thermoplastischer Polymere ist stark von der vorliegenden Faserorientierung abhängig. Für die Auslegung von Bauteilen aus derartigen Materialien werden in der Struktursimulation Materialmodelle eingesetzt. Diese Modelle werden über experimentell ermittelte Kennwerte parametrisiert und validiert. Hierzu benötigt man Probekörper mit möglichst homogener und hoher Faserorientierung. Die Herstellung von diesen Probekörpern sowie der Einfluss der fräsinduzierten Oberflächenrauigkeit auf die mechanischen Eigenschaften während der Probenpräparation stellen die zentralen Forschungsfragen der vorliegenden Arbeit dar. Es werden verschiedene Ansätze für eine Prüfplatte mit homogener und hoher Faserorientierung erarbeitet, um die für Spritzgussplatten typische dreischichtige Morphologie zu vermeiden. Das erfolgversprechendste Konzept wird in einer Spritzgussform umgesetzt. Mikrocomputertomographische Analysen weisen für Polybutylenterephthalat und Polyamid 66 je mit 30 Gewichtsprozent Kurzglasfasern verstärkt eine homogene und hohe Faserorientierung nach. Um den Einfluss der fräsinduzierten Oberflächenrauigkeit auf die mechanischen Kennwerte zu untersuchen, werden Probekörper aus den hochorientierten Prüfplatten heraus gefräst. Hierbei werden über die Drehzahl und den Vorschub gezielt unterschiedliche Rauigkeiten eingestellt. Die Proben mit verschiedenen Faserorientierungen werden unter einachsiger Zugbelastung bei Raumtemperatur und -10 °C betrachtet. Für beide Temperaturen zeigt die systematische Analyse keinen signifikanten Einfluss der Oberflächenrauigkeit auf die mechanischen Kennwerte. Folglich reagieren die mechanischen Kennwerte mit dieser Versuchsmethode unempfindlich auf die Fräsparameter. Es konnte damit nachgewiesen werden, dass die Probenpräparation mittels Fräsen zur Herstellung von mechanischen Probekörpern für die verwendeten Materialien geeignet ist.
EMV-Untersuchungen für die Qualifizierung von elektrischen Orbitalantrieben für Satelliten
(2024) Rover, Yannik
Bei dieser Arbeit handelt es sich um eine kumulative Dissertation. Die Forschungsarbeit beschäftigt sich mit Untersuchungen der elektromagnetischen Verträglichkeit (EMV) an Orbitalantrieben für Satelliten. Grundlage dieser Arbeit ist die voranschreitende kommerzielle Nutzung von elektrischen Raumfahrtantrieben. Hierfür steht ein Radiofrequenz-Ionentriebwerk (RIT) als Stellvertreter der elektrostatischen Triebwerke zur Verfügung. Veröffentlichungen in themenspezifischen Journalen dokumentieren die durchgeführten Arbeiten und zeigen die gewonnenen Erkenntnisse. Dabei handelt es sich um die Generierung und Erweiterung von Simulationsmodellen für das Triebwerk, inklusive Ionenstrahl, wie auch dessen Peripheriekomponenten. Weiterhin sind der Aufbau und die Inbetriebnahme einer halb-reflexionsfreien Absorberkammer mit Vakuumtank beschrieben. Diese Ausarbeitung dient der Einordnung und Zusammenfassung der veröffentlichten Beiträge, durch welche ein umfassendes Wissen innerhalb der Themenfelder entstanden ist und somit wiederum neue Arbeiten ermöglicht werden. Die Einflüsse auf die EMV eines Triebwerks, durch einen Radiofrequenz-Generator sowie einen Ionenstrahl, sind durch Simulationen nun vorhersagbar und werden in weiteren Schritten zusammengeführt, um ein umfassendes Modell zu erhalten. Die optimierte halb-reflexionsfreie Absorberkammer eliminiert die Schwächen bestehender, vergleichbarer Testeinrichtungen und ermöglicht aussagekräftige Analysen.
Finding the Comfort Zone: Bacteria-Surface Interaction in Microbial Fuel Cells
(2022) Frühauf-Wyllie, Hanna Marianne
In microbial fuel cells (MFC) (biological) waste can be converted to electrical power and thus contribute to the development of a sustainable circular economy. The core of any MFC are exoelectric bacteria that oxidise organic substrates and transfer resulting electrons to an electrode as terminal electron acceptor. Exoelectric bacteria that rely on direct electron transfer require immediate contact with the electrode, which gives the physico-chemical properties of the electrode surface particular importance. This is the case for the MFC model organism Geobacter sulfurreducens that was used in this work. The aim was to improve bacteria-surface interaction by adapting the electrode surface characteristics in order to increase MFC efficiency. The bacterial adhesion to a surface depends on a variety of physico-chemical factors; the one in focus for this work was surface charge. Layer-by-layer coating with differently charged polyelectrolytes was used to modify the surface charge of MFC anodes. Subsequently, the effect on selected MFC performance indicators was assessed. MFC with indium tin oxide (ITO) anodes (polarisation +0.1 V vs. Standard Hydrogen Electrode) were used to define reference values against which the results on coated anodes could be compared. Anode surface charge influenced all analysed performance indicators and the results were subject to a general correlation: The thicker and more viable the biofilm, the higher were current density and coulombic efficiency, and the shorter was the start-up phase. However, none of the coatings significantly improved the performance compared to the non-coated electrodes (maximum current density on ITO: 399 μAcm-2 ± 24% (n = 3), maximum current density with negatively charged polystyrene sulfonate as terminating layer: 456 and 377 μAcm-2, respectively). The other coatings resulted in poorer performance. This contradicted the hypothesis accepted in literature that a positive surface charge is generally beneficial for bacterial adhesion. To gain a better understanding of the initial phase of biofilm formation, an electrochemical flow cell that can be operated under a confocal laser scanning microscope was used. The initially desired application of the flow cell (in vivo biofilm analysis) failed due to a fluorescent G. sulfurreducens strain that a) did not form a G. sulfurreducens-typical biofilm and b) did not develop sufficient fluorescence for microscopic analysis under anaerobic conditions. However, the results emphasised that the optimisation of MFC is difficult due to the high number of factors that influence the performance. The interaction between electrode surface coating, pH and salinity of the medium and any surface-active macromolecules released by bacteria will add on to the sole effect of the surface modification and complicates the isolated analysis of influencing factors. Also, the comparison to existing MFC improvements through surface modification is made difficult by the multivariate system the MFC is. Nevertheless, monitoring of the initial bacteria-surface interaction in vivo and recording the corresponding current response is a promising strategy when aiming to improve MFC anode material and can hopefully push this promising technology another step ahead.
Identification of critical process parameters for the downstream processing of cell culture-derived Orf virus particles
(2023-12-04) Eilts, Friederike
The COVID-19 (coronavirus disease 2019) pandemic has shown the importance of ready-at-hand- tools to bring pharmaceutical products to market. This includes the availability of plat-forms to target new diseases as well as their production process. Such a platform could be available with the Orf virus (ORFV), a promising viral vector with applications as gene thera-peutic, antiviral, oncolytic, immunomodulatory, and vaccination agent. However, production processes for the ORFV are not established yet. A recent publication on a lab-scale purifica-tion process of the ORFV suggested the use of the steric exclusion chromatography (SXC) with high infectious virus yields and impurity removal. This rather new method is closely related to the conventional polyethylene glycol (PEG) precipitation, however, including a po-rous chromatographic media in the process. By preferential and steric exclusion mechanisms, the precipitates accrete inside the stationary phase and are retained. Several critical process parameters of the method are known and easily controlled, i.e., the PEG composition, the buffer composition as well as the pH and the ionic strength, the mixing technique prior to column loading, the flow rate, and the stationary phase. Nevertheless, high variability be-tween different runs is frequently observed. In this work, three unexplored parameters for ORFV processing with SXC were identified: the pore size of the stationary phase, the incuba-tion time and mixing strategy of the PEG / ORFV solution, and the addition of salts. Here, small pore sizes and long incubation times induced filtration effects, which cause pressure surges. The pressure is often a constraint in SXC application due to the high viscosity of the PEG. Therefore, a reduction of the PEG concentration while maintaining high yields is desira-ble. This could be achieved by adding different chaotropic ions to the PEG/ORFV mixture as proven for Mg2+. Furthermore, characterization of degrading and stabilizing conditions on the infectivity of the new platform virus was rarely published. In this work, this gap was addressed by an extensive study of mechanical, chemical, and thermal stress conditions on ORFV infec-tivity. In summary, the ORFV is extremely robust against degradation under conditions rou-tinely applied for pharmaceutical virus production. Nevertheless, care should be taken with heat and the implementation of ultrasonication in the production process. Generally, the ORFV should be stored under refrigerated conditions (4 °C), although the virus showed no substantial loss of infectivity if stored at 37 °C for two days or frozen. With regards to ion typically applied in purification processes, only ammonium salts should be avoided. Further stabilization is expected by the addition of proteins, sugars, and some amino acids. Based on these findings, a formulation buffer for ORFV storage with 1 % recombinant human serum albumin and 5 % sucrose was proposed. In the case of extended storage under heated condi-tions, albumin can be substituted with arginine. In conclusion, this comprehensive study of preferable process and storage conditions and critical process parameters of the SXC for the ORFV can inform future scale-up options to successfully implement SXC in pharmaceutical ORFV production processes with high infectious titer recoveries.
Investigation of hot plume impact on space launch vehicle aft-body flows
(2022) Schumann, Jan-Erik
The transonic flow around generic space launch vehicle aft-body geometries is investigated numerically using hybrid RANS-LES methods. A sensitivity study shows that the qualitative results are mostly insensitive to changes in grid resolution and numerical settings, but certain aspects such as circumferential grid resolution and LES filter length definition can affect the obtained quantitative results. It is further found that the presence of a plume leads to the appearance of an additional spectral contribution in the flow. The main investigations showed that changing the plume properties leads to a downstream shift of the reattachment location due to the stronger acceleration of the external flow. An increased wall temperature leads to a similar shift that is attributed to the slower development of turbulent structures in higher temperature shear layers. As a consequence the pressure fluctuations and the nozzle forces are reduced as well. However, the underlying flow phenomena are unaffected by the plume properties and wall temperature, with the exception of an additional spectral feature in the pressure fluctuations due to the interaction of shear layer movement and nozzle flow plume separation once the reattachment occurs mostly beyond the nozzle lip. A shorter nozzle strongly affects the recirculation region shape, pressure fluctuations and nozzle forces and does not show the observed flow phenomena observed for the longer nozzle configuration.
Novel Double Cantilever Beam experiments and related evaluation methods to investigate the fracture behaviour of adhesive layers in modes I and III
(2022) Schrader, Peer
This cumulative dissertation consists of four manuscripts dealing with the fracture mechanics testing of adhesive layers in pure loading modes I and III. Different Double Cantilever Beam (DCB) test specimens are used to investigate the energy release rate and cohesive traction of rigid, epoxy-based and soft, rubber-based adhesive systems under different boundary conditions. New test setups and evaluation methods are developed to gain a better understanding of the fracture behaviour of these adhesive joints and to investigate the influence of different evaluation methods on the results. In order to investigate the fracture behaviour of epoxy-based bonded joints, the Out-of-plane Loaded Double Cantilever Beam (ODCB) test was extended to higher loading rates, which, with the help of a local strain measurement, allowed the determination of fracture energy and cohesive traction in mode III at increased loading rates for the first time. However, it became apparent that ODCB tests, which have to be carried out in a bi-axial testing machine, are probably not suitable for testing under impact. Hence, uni-axial test rigs were developed to enable mode III testing of such adhesive joints. These were carried out quasi-statically to investigate the influence of different evaluation methods in more detail. The rate-dependent fracture behaviour of soft, rubber-like adhesives was also investigated, which includes both a creep load in pure mode I and an investigation at increased loading rates in modes I and III. For the investigation under creep loading, a method is proposed which allows the control of the tests on constant J-integral. In addition, the influence of the material behaviour within the process zone is examined in more detail, as this influences both the creep behaviour and the cohesive stresses and their evaluation methods. For the determination of the cohesive traction, a new method is developed within the scope of the work, which is based on the strain measurement along with the adherends and thereby enables a determination of the dependence of the cohesive stresses on the loading rate along the entire adhesive layer. A comparison with the conventional method of determining the cohesive traction from the derivative of the energy release rate with respect to the crack opening displacement showed that the currently applied method may deliver inadequate results. The results of this work thus extend the state of the state of research and in doing so demonstrate new methods that can be used in DCB tests in modes I and III to provide new insights into the fracture behaviour of adhesively bonded joints.
Ein Radiofrequenz-Generator mit Parallelschwingkreis für den effizienten Betrieb eines Radiofrequenz-Ionentriebwerks
(2022) Junker, Jan Erik
Die vorliegende Arbeit thematisiert die Entwicklung eines Radiofrequenz-Generators mit Parallelschwingkreis, der zur induktiven Energieeinkopplung bei Radiofrequenz-Ionentriebwerken verwendet wird. Dazu erzeugt der Generator einen hochfrequenten sinusförmigen Strom, mit dem die Induktionsspule des Triebwerks beaufschlagt wird. Um den Betrieb des Generators mit einem hohen Wirkungsgrad gewährleisten zu können, kommt ein Regelungskonzept zum Einsatz, welches die Schaltfrequenz der Halbleiterschalter hochdynamisch auf die entsprechende Last anpasst. Neben dem Schaltungs- und Regelungskonzept werden Simulationsergebnisse und experimentelle Untersuchungen am Triebwerk vorgestellt. Dabei wird der entwickelte Prototyp mit einem Radiofrequenz-Generator mit Serienschwingkreis verglichen.
Steric exclusion chromatography: Advancement of a laboratory-based platform technology into a key component of viral vector and vaccine production processes
(2021) Lothert, Keven
Biological macromolecules, such as viruses, virus-like particles or extracellular vesicles represent an important and permanently growing element in the biopharmaceutical industry. These macromolecules applied as pharmaceutical products have proven suitable for prophylactic or therapeutic vaccination, oncolytic therapy or gene transfer, to name only a few. Beside the challenge of producing sufficient amounts of product, its purification during the downstream processing, for example from cell culture harvests, is of major importance to allow a safe and efficient application. A large variety of particles differing in their physicochemical properties, possibly affecting their stability and their interaction with surrounding molecules, requires multifaceted processing strategies. For this reason, purification process development can be a time and cost consuming element, which emphasizes the benefits of flexible techniques that can be applied to a variety of different products. Over the past ten years the steric exclusion chromatography (SXC) was introduced as a possible platform technique for a fast and effective purification of macromolecules and nanoplexes such as large proteins, bacteriophages, and viruses. The technique was derived from the principle of polymer induced crowding mechanisms, that are already known for several decades. Although the method highly depends on the size of the target product, all the influencing process parameters and mechanisms of the method are not yet thoroughly understood. The SXC has been applied as an independent process unit operation but has not been implemented in large scale biopharmaceutical production. The latter can be mainly attributed to the novelty of the principle, the lack of appropriate stationary phases suitable for industrial production, and an inherent skepticism against new approaches compared to established and regulatory accepted procedures. This work aims to bridge laboratory applications of the SXC with scaled approaches of up to 200 L by suggesting possible processing schemes employing that technique as a major purification backbone. Simultaneously, these studies provide further insight into critical process parameters to allow for an improvement of the mechanistic understanding concerning the method itself. In the chapters of this work the successive implementation of the SXC into complete downstream processing schemes for viral vectors and vaccines is shown. While Chapter 1 gives an overview to introduce the importance of improving and extending the existing portfolio of purification procedures, it also introduces the benefits of applying platform technologies. Particularly the principle and advantages of the SXC are described as well as the scope of this thesis is outlined. Chapter 2 summarizes the general approach for applying and optimizing the SXC using the baculovirus vector as a model and Chapter 3 shows a screening comparison of the SXC against and in combination with well-known purification techniques. Afterwards, in Chapter 4, the platform applicability in complete processing schemes is shown for Hepatitis C and Orf virus purification targeting potential applications in human or veterinary medicine. Finally, Chapter 5 gives a short summary, including an outlook on future prospects and remaining hurdles.
Sustainable use of green waste and CO2 in (electro-)biotechnology
(2024) Langsdorf, Alexander
In this work, material recycling methods for green waste in (electro)biotechnology and the bioelectrochemical use of CO2 from flue gas were investigated. The overall aim was to find alternatives to conventional recycling of green waste and ways to reduce CO2 emissions. Initially, it was investigated whether grass clippings, a main component of green waste, can serve as a substrate for fermentation. For this purpose, grass clippings were homogenized and the solids separated. The juice was examined as a medium additive and as the sole medium for the cultivation of Cupriavidus necator pKR-hum. Different sterilization methods for the grass juice and different concentrations as an additive in the fermentation medium were tested. Cupriavidus necator pKR-hum could be successfully cultivated with autoclaved grass juice as the sole growth medium without further additives. A growth rate of 0.43 h-1 was achieved on the grass medium compared to 0.50 h-1 on a conventional LB medium. In addition, 2 mg L-1 of the terpene α-humulene could be produced on the grass medium. As a further utilization method, the extraction of industrially relevant enzymes from green waste was investigated. For this purpose, the common lawn grasses Lolium perenne and Festuca arundinacea were cultivated in the laboratory and a purification scheme for peroxidases was developed. The purified peroxidases were characterized with regard to reaction optima and kinetics and used for the oxidation of the phenolic substances phenol, m-cresol, and 2,4-dichlorophenol for wastewater treatment. The peroxidases of both grasses showed efficient conversion of the substrate 2,4-dichlorophenol. Subsequently, the experiments were repeated with crude extracts of the individual grasses and with a crude extract of real grass clippings. With the crude extract of the real grass clippings, more than 95% of 0.5 mM 2,4-dichlorophenol could be converted within 20 min. Finally, the use of green waste as starting material for electrodes for bioelectrochemical systems was investigated. Grass clippings were carbonized via hydrothermal carbonization and pyrolysis. Electrodes were manufactured from the resulting biochar using a binder and a metallic carrier. Biochar and electrodes were characterized in terms of materials science and electrochemistry. The electrodes were then used in microbial electrosynthesis (MES) with Cupriavidus necator H16 PHB-4 and in a microbial fuel cell (MFC) with Geobacter sulfurreducens and compared with conventional electrodes. As expected, the manufactured electrodes were less suitable for water electrolysis in the MES than conventional metal electrodes. However, the manufactured electrodes showed a similar performance to commercial graphite electrodes in the MFC. In addition, long-term operation of the MFC for more than six weeks could be realized. With regard to real-life application in an industrial environment, the MES was carried out in a cogeneration plant to investigate flue gas as a possible source of CO2. For this purpose, Cupriavidus necator H16 was used to produce the bioplastic polyhydroxybutyrate (PHB) in the MES. The MES was carried out and compared in the laboratory with a clean gas mixture and in the cogeneration plant with real flue gas. The use of flue gas had no detectable effect on either microbial growth or PHB production in comparison to the experiments in the laboratory. In the cogeneration plant, 333 ± 44 mg L-1 PHB could be produced with the MES at a proportion of 43 ± 3% of the dry cell mass. The results demonstrate alternative recycling methods for green waste and CO2 from flue gas, which can contribute to a reduction in the use of fossil resources and CO2 emissions. Overall, this work can support a shift towards a bioeconomy and a circular economy.

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Auflistung Dissertationen/Habilitationen nach Auflistung nach Fachbereich/Einrichtung "PZI Promotionszentrum für Ingenieurwissenschaften"

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