Veiter, L. (2019). Morphological PAT tools for filamentous bioprocesses : monitoring and control enhanced for fungal morphology [Dissertation, Technische Universität Wien]. reposiTUm. https://doi.org/10.34726/hss.2019.43608
E166 - Institut für Verfahrenstechnik, Umwelttechnik und technische Biowissenschaften
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Date (published):
2019
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Number of Pages:
171
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Keywords:
Penicillium chrysogenum; Flow Cytometry; Penicillin; Viability; Morphology; Process monitoring; active biomass; filamentous fungi; pellets; image analysis
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Abstract:
Biomass is one of the most essential process variables in bioprocesses. Process performance, process controls strategies and productivity in filamentous processes highly depend on cellular aspects, such as morphological elements of the biomass which calls for a segregated view of biomass. In filamentous bioprocesses, quality of the biomass is of prime importance. Hereby one strives to ensure high biomass viability and productivity. Filamentous fungi display a large variety of morphological forms in submerged cultures. These range from dispersed hyphae to denser hyphal aggregates, the so-called pellets. Morphology, viability and productivity are tightly interlinked. Depending on the objective function of the bioprocess, different characteristics of morphology are favourable and need to be quantified accordingly. Morphology and viability can be determined via a variety of methods, each limited to specific areas of application. The most common method to characterize morphology is image analysis based on microscopy, which is work intensive and time consuming. Typical methods to determine viable biomass encompass di-electric spectroscopy or staining reagents, which is troublesome regarding the complex fungal morphology. Therefore, our objective was to develop several alternative, robust and statistically sound methods based on flow cytometry and fluorescent staining for at-line application capable of assessment of morphology and viability. Furthermore, novel morphological characteristics describing pellet biomass are assessable via these methods: ‘pellet compactness and ‘viable pellet layer. Additionally, spatially resolved mass spectrometry was used in a novel technique to study metabolism and productive zones in fungal pellets. Our superior goal was to apply said analytical methods to study novel morphological responses and use this knowledge to determine a process design space in fermentation ensuring optimal morphology, viability and productivity. To achieve this, we employed and developed additional control strategies to regulate the growth rate or specific substrate uptake during Penicillium chrysogenum fermentations. In a design of experiments (DOE) approach, fermentation factors power input, dissolved oxygen concentration and specific substrate uptake rate were varied to feed a data-driven model including novel morphological responses. Thereby an optimised process design could be obtained which resulted in enhanced biomass viability and specific productivity. We envision that the methods compiled in the comprehensive ‘Analytics and ‘Control chapters of this Thesis will serve as process analytical technology (PAT) tools specifically enhanced for complex fungal morphology and providing novel morphological descriptors. Additionally, we have successfully tested our methodology on other agglomerate-forming organisms like yeast to demonstrate versatility and transferability.