Browsing by Subject "Hefeartige Pilze"

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Bioethanol production from lignocellulosic biomass
(2023) Hoppert, Luis; Kölling, Ralf
The aim of this thesis was to develop a high gravity second-generation bioethanol process and investigate the effects of a high solid loading. The insights gained from the initial experiments helped to understand the underlying mechanism behind the limitations of a high solid loading. Based on these findings, strategies were developed to overcome these limitations.
Detektion von Schadhefen in Wein mittels mit Flusszytometrie analysierter in situ Hybridisierung (Flow-FISH)
(2021) Willberger, Ilka Nadine; Scharfenberger-Schmeer, Maren
In oenological practice, mostly unpasteurised grape musts are used. This leads to an increased introduction of non-saccharomyces, which can have a lasting effect on the fermentation process. Disturbances in the fermentation process are usually only detected in practice on the basis of abnormalities in selected parameters such as sugar content or temperature or the occurrence of off-flavours. The fermenting yeast population may already be so affected at this point that intervention in the fermentation process can no longer prevent the occurrence of off-flavours in the end product or incomplete fermentation. With the help of flow cytometry, an efficient method using FISH (Fluorescence In Situ Hybridisation) was developed to detect and quantify the common representatives of the fermentation population such as Sacchoromyces cerevisiae and the harmful yeast population such as Hanseniaspora uvarum, Dekkera bruxellensis and Pichia anomala in the course of fermentation. Rapid detection enables countermeasures to be taken in good time before a harmful yeast population can have too great of an influence on the course of fermentation and the metabolites formed. Flow-FISH was established with pure cultures from strain collections in defined medium (YPD) and pasteurised white grape must. Samples are extracted and fixated directly from fermentation mixtures. For hybridisation, 18S- and 26S-rRNA probes with FITC-labelling are used. For the evaluation of the flowcytometric data, the Overton-subtraction method is used in this work. This allows a more accurate assessment of the hybridised cell population than the usual setting of a marker. For this purpose, an effective negative control with complementary sequence to the universal eukaryote probe (Euk516) is introduced. Subsequently, the method already known from the literature was optimised with regard to hybridisation conditions and cell fixation and thus adapted to the requirements of a quantitative flow cytometric analysis. With fixation in formaldehyde or in ethanol, fixation methods were developed that fulfil the requirements of both rapid and reliable fixation in the laboratory and rapid fixation in the cellar, if transport to the laboratory is not possible in a timely manner.Helper probes were designed to increase the fluorescence intensity. They are unlabelled and bind in the direct proximity of the specific probe. In all yeast species investigated, S. cerevisiae, H. uvarum, D. bruxellensis and P. anomala, the fluorescence intensity can be considerably increased by using the helper probes. In the case of D. bruxellensis and P. anomala, detection is only possible with the use of the helper probes. The helper probes allow the Flow-FISH assay to be used in a broader growth range of the yeast culture. Without helper probes, quantitative detection is limited to the middle logarithmic growth phase. With helper probes, hybridised cells can be reliably detected starting in the early logarithmic growth phase up until the stationary phase. This covers the critical phase of fermentative activity so that increasing contamination can be detected in the fermentation.The specificity of the probes is given. In part, there are slightly increased fluorescence intensities compared to the negative control, especially with the D. bruxellensis probe combination and non-specific yeasts, which can probably be attributed to increased binding due to the composition of this probe combination.The Flow-FISH assay is also reliable in mixtures of different yeast species and up to a cell count of 10³ cells / ml in the initial fermentation. This detection limit is also achieved by other methods in molecular biology for yeast detection. In contrast to most of these methods, Flow-FISH can also quantify the number of yeasts present. Additionally the use of the flow cytometer offers a simple variant to determine the total cell count of all yeasts in the fermentation. The detection limit of Flow-FISH allows detection before the damage threshold values of the yeasts examined are reached. The Flow-FISH method presented in this dissertation can also be applied to other yeast strains, some of which also originate from wild isolates. A transferability to native fermentations from oenological practice is given. It was possible to examine both spontaneous fermentations and inoculated fermentations in practice fermentations in steel tanks for their yeast population composition and to follow their development in the course of fermentation. Due to the use of flow cytometry and the helper probes and negative control used in this dissertation, the optimised Flow-FISH assay offers a stable basis for the continued development of a test system for use in oenological practice.
Influence of the newly identified Mos10 interaction partner Vps68on ESCRT-III function
(2021) Alsleben, Sören; Kölling, Ralf
The endosomal sorting complex required for transport (ESCRT) is a part of the heteromeric complex machinery consisting of ESCRT-0, -I, -II, and -III ensuring functional protein traffic of endocytic and biosynthetic cargo. Stepwise sorting of labeled cargo material inside the lumen of the endosome by invagination and abscission of the endosomal membrane to form intraluminal vesicles (ILV’s) is mediated by the ESCRT-III complex. The complex consists of eight members of which Vps20, Snf7, Vps2, and Vps24 are considered ESCRT-III essential subunits, and Chm7, Did2, Ist1, and Mos10/Vps60 are commonly labeled as complex associated proteins. The correct interplay between the proteins ensures cargo sorting into the MVB (multivesicular body) pathway and transport from the late endosome into the vacuolar lumen for degradation. Besides the initial function of vacuolar protein sorting (vps), the complex is involved in a multitude of cellular processes like cell abscission, virus budding, autophagy, and remaining nuclear envelope integrity. The step-wise assembly of the ESCRT-III complex is mediated after the cascade-like ESCRT-0 to ESCRT-II complex formation at the membrane budding site, collecting cargo protein for invagination into the endosomal lumen. ESCRT-III Vps20 is recruited to the membrane by the ESCRT-II member Vps25, then nucleating Snf7 association and oligomerization. Additional assembly of ESCRT-III members like Vps24 and Vps2 further drives membrane bending away from the cytosol to the final abscission event, before being recycled back to cytosolic monomers by Vps4. Although Mos10 has been implicated in the recycling step of the ESCRT-III units by interacting with the Vps4/Vta1 complex, the protein’s function remains poorly characterized. This thesis tried to find new insights in Mos10 functionality by finding yet uncharacterized interacting partners, thus connecting the protein to new putative non-endosomal functions or understanding its role in the established ESCRT-III complex. For this purpose, a series of crosslinking experiments with tagged variants of Mos10 were performed. Purification was achieved by IMAC (Immobilized Metal Ion Affinity Chromatography) after adding a poly-his sequence to the protein and by immunoprecipitation of sfGFP tagged Mos10. Both methods revealed a multitude of putative Mos10 interacting partners by MS analysis to be further reduced by applying the SILAC (stable isotope labeling with amino acids in cell culture) technique. After selecting possible Mos10 interacting partners, IP and Co-IP experiments of tagged candidate variants were used to identify an interaction between the two proteins. An interaction between Mos10-6His and Vps68-13myc besides native Mos10 and Vps68-fGFP could be verified by purification of Vps68 and co-precipitating Mos10. The influence of Vps68 on the assembly and composition of the ESCRT-III complex was examined. After Vps68 depletion, an enrichment of the core subunits Snf7, Vps2, and Vps24 in the complex was detected with a reduced number of Did2, Ist1, and Mos10 molecules. Thus, it appears that ESCRT-III disassembly is blocked in ∆vps68 mutant. The influence of VPS68 deletion on the intracellular localization of ESCRT-III proteins was examined by fluorescence microscopy with sfGFP-tagged variants. While the localization of most ESCRT-III proteins was not significantly altered, a marked relocalization was observed for Mos10. In wildtype, Mos10-sfGFP was localized at the vacuolar membrane, while in ∆vps68 it was dispersed into vesicular structures enriched at the cell cortex. Further, the impact of VPS68 deletion on the sorting of the endocytic cargo protein Ste6 was investigated. By cycloheximide chase experiments, it could be shown that Ste6 is strongly stabilized in a ∆vps68 mutant. This indicates that the transport of the protein to the yeast vacuole for degradation is blocked. The ∆vps68 block in endocytic trafficking was compared with other mutants of the vps-pathway, whose site of action has been established. These experiments show that the VPS68 deletion neither leads to a class D phenotype, as in ∆vps21, nor to a class E phenotype, as in ∆snf7. The Ste6-GFP distribution in the ∆vps68 mutant rather resembles wildtype with more pronounced accumulation of endosomal dots. The data taken together suggest that Vps68 acts after the formation of the ESCRT-III complex and is required for cargo delivery from the late endosome to the vacuolar lumen.
UV-C-Behandlung von Traubenmost zur Inaktivierung von Mikroorganismen
(2018) Diesler, Kathrin; Scharfenberger-Schmeer, Maren
The development of new preservation process techniques to protect ingredients and maintain a high quality standard is always a main goal in the food industry. In course of this, microbial safety has top priority. UV-C technology is a modern, non-thermal process with high efficiency. It has been used for sterilization and treatment of drinking water for many years. Also, ultraviolet radiation for disinfection purposes is already being used in other areas of food production. To what extent this method can be successfully applied in the field of grape must production, will be investigated in this dissertation. For this purpose, several yeasts and bacteria, relevant in this area, were examined for their inactivation potential by UV-C treatment. To ensure the best possible microbial inactivation in must, it is essential to determine an ideal treatment dose for both yeasts and bacteria. The results have confirmed that bacteria are far more sensitive to UV C treatment, than yeasts. It was also shown that there are major differences in UV-C stability within the seven yeast species and six bacteria species used in this study. The analyses have identified Metschnikowia pulcherrima and Acetobacter aceti as the most UV-C stable and Brettanomyces custerianus and Pediococcus sp. as the most sensitive organisms. Furthermore, three morphologically different Brettanomyces strains were used to show that there are also strain-specific variances in the response to UV-C treatment. Using Saccharomyces cerevisiae as an example, a potential formation of UV-C resistance was also ruled out. For this purpose, yeast cells were exposed to a dose, that did not result in complete inactivation. The surviving cells were cultured and retreated. Even after repeating this process eight times, no change in the UV-C response of the yeast cells could be detected. For the application of UV-C technology in the juice and wine industry it has to be ensured, that microorganisms are killed directly and their enzymatic activities are directly inhibited. Yeasts and bacteria could further convert sugar to alcohol or form unwanted metabolic byproducts. Therefore, the enzymatic activity after the initial treatment and during the inactivation process of Saccharomyces cerevisiae was analyzed in more detail. HPLC was used to determine the content of glucose, fructose and ethanol. No enzymatic activity could be detected in the UV-C treated samples from the moment after the initial UV-C treatment, up to the day of complete destruction. However, the effectiveness of UV-C treatment of must and wine cannot be attributed solely to the responsiveness of the various microorganisms. Other product parameters such as grape variety, turbidity and optical density also play a decisive role. In this context, four different musts with different optical density and turbidity were treated and the inactivation kinetics of Saccharomyces cerevisiae were compared. In this work it could be proved, that with an increasing optical density and a higher turbidity, the efficiency of the UV-C treatment in must decreases strongly. The success of a treatment is also directly dependent on the initial contamination rate of the product. Tests with different starting cell numbers have shown, that the required inactivation dose also has to be increased, as the number of cells increases. In the winemaking process, however, not only yeasts and bacteria can be a potential source of danger. The fungal infection of grapes by Botrytis cinerea also carries a high risk. The polyphenol oxidase laccase, produced by the fungus, damages ingredients and leads to a colour change in must and wine. In the investigations it could be proven, that it is possible to strongly reduce or completely inactivate the enzymatic activity in Botrytis infected must, depending on the starting concentration. In summary, UV-C technology represents an effective alternative and extension for current oenological practice. It offers the possibility to inactivate a large number of wine relevant microorganisms without causing resistance. In addition, this work has created a new framework for the application of must specific parameters. The results for the inactivation of the enzyme laccase are also proved to be extremely promising.