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Knufia petricola is a black fungus that colonizes sun-exposed surfaces as extreme and oligotrophic environments. As ecologically important heterotrophs and biofilm-formers on human-made surfaces, black fungi form one of the most resistant groups of biodeteriorating organisms. Due to its moderate growth rate in axenic culture and available protocols for its transformation and CRISPR/Cas9-mediated genome editing, K. petricola is used for studying the morpho-physiological adaptations shared by extremophilic and extremotolerant black fungi. In this study, the bacteria-derived tetracycline (TET)-dependent promoter (Tet-on) system was implemented to enable controllable gene expression in K. petricola. The functionality i.e., the dose-dependent inducibility of TET-regulated constructs was investigated by using GFP fluorescence, pigment synthesis(melanin and carotenoids) and restored uracil prototrophy as reporters. The newly generated cloning vectors containing the Tet-on construct, and the validated sites in the K. petricola genome for color-selectable or neutral insertion of expression constructs complete the reverse genetics toolbox. One or multiple genes can be expressed on demand from different genomic loci or from a single construct by using 2A self-cleaving peptides, e.g., for localizing proteins and protein complexes in the K. petricola cell or for using K. petricola as host for the expression of heterologous genes.
Der Band 5 der N.i.Ke.-Schriftenreihe präsentiert den aktuellen Stand materialwissenschaftlicher und bildgebender Verfahren, die bei der Untersuchung von Zeichnungen Anwendung finden. Im ersten Teil der Publikation werden die klassischen Zeichnungsträger und Zeichnungsmaterialien vorgestellt. Der mittlere Teil der Publikation beschreibt die relevanten materialwissenschaftlichen und bildgebenden Verfahren. Der letzte Teil diskutiert anhand einiger Fallbeispiele die Möglichkeiten und Grenzen der Methoden.
Antimicrobial resistance (AMR) is a global health problem. It is well known that antibiotics can drive evolutionary processes that underlie antimicrobial resistance (AMR) evolution and spread in clinical and environmental settings. In contrast, less is known about the effects of antimicrobial substances that are used as biocides (i.e. disinfectants and preservatives) on AMR evolution and spread. Biocides are present in various settings, interacting with diverse microbial communities. Therefore, it is crucial to evaluate their role in the evolution and dissemination of antimicrobial resistance. Biocides occur in a wide range of concentrations in various environmental settings. By examining how the various concentrations affect selection mechanisms, we gain insights into potential developments related to antimicrobial resistance. The aim of this PhD thesis is to investigate the effects of biocides on processes underlying resistance evolution. Specifically, the work focused on key mechanisms for resistance spread, resistance evolution, and the effect of selection pressures on evolved resistance mechanisms. The thesis is structured around three major objectives: (i) to determine the effect of biocides on the evolution of resistance by affecting the rate of occurrence of de novo mutations, (ii) to determine the effect of biocides on the spread of resistance genes by modifying the rate of horizontal gene transfer (HGT) processes, and (iii) to investigate the selective drivers of the emergence of antimicrobial resistance in adaptive laboratory evolution (ALE) experiments. De-novo mutations are spontaneous mutations that occur at a certain rate in microorganisms. The effect of biocides at subinhibitory environmentally relevant concentrations on the mutation rate in Acinetobacer baylyi, Bacillus subtilis and Escherichia coli was assessed with the fluctuation assay. The results showed that biocides affected mutation rates in a species and substance dependent matter. The bisbiguanide chlorhexidine digluconate, the quaternary ammonium compound didecyldimethylammonium chloride, the metal copper, the pyrethroid-insecticide permethrin, and the azole-fungicide propiconazole increase mutation rates in E. coli, whereas no increases were identified for B. subtilis and A. baylyi. Horizontal gene transfer refers to diverse mechanisms that mediate the transfer of mobile genetic elements between microorganisms. This work focused on conjugation and transformation. Conjugation is a process whereby a conjugative plasmid is transferred from a donor cell to a recipient cell. Transformation is a process whereby exogenous donor DNA is taken up into a recipient cell and integrated into the recipient’s’ genome. The effects of subinhibitory environmentally relevant biocide concentrations on the conjugation rate of E. coli and the transformation rate of the naturally competent organisms A. baylyi in were assessed. The results showed that benzalkonium chloride (BAC), chlorhexidine and permethrin increased conjugation in E. coli, while none of the biocides increased transformation rates in A. baylyi. To further understand the molecular mechanisms underlying the effects on mutation and conjugation rates, I investigated the induction of the RpoS-mediated general stress and the RecA-linked SOS response upon biocide exposure. The results show a link between the general stress and the SOS response with increased rates of mutation and conjugation, but not for all biocides. One major approach to study the evolutionary response of bacteria to antimicrobials are ALE experiments with growth at subinhibitory concentrations linked to serial subculturing over many generations. Such experiments have been used to study resistance evolution to antibiotics and biocides. However, previous work showed that adaptation to biocide stress may be mediated by different evolutionary drivers. Here, I investigated the contributions of evolution for increased survival as opposed to improved growth in ALE experiments with E. coli exposed to subinhibitory BAC concentrations. Two distinct evolutionary treatments selecting for survival only or survival and growth led to specific evolutionary adaptations apparent in the phenotypes and genotypes of the evolved populations. Populations growing in the presence of BAC evolved increased fitness in the presence of BAC associated with higher resistance to BAC and cross-resistance to antibiotics, while this was not the case for populations evolving for increased survival only. Genotypic characterization by whole genome sequencing of the evolved populations revealed parallelism in mutated genes among replicate populations and distinct differences across treatments. Treatments selecting for survival and growth showed mutations in stress response related genes (hslO and tufA), while selection for survival led to mutations in genes for metabolic regulation (cyaA) and cellular structure (flagella fliJ). In summary, this thesis shows that biocides affect AMR evolution and emphasizes the importance of understanding of how biocides impact the molecular and evolutionary process that underlie AMR evolution.
Advanced light weight applications like aircrafts and wind turbine blades are made of fibre reinforced plastics (FRP) with continuous fibre reinforcement and must withstand a high thermo-mechanical cyclic loading. The quality of the fibre matrix interface has a high impact on the fatigue life and was continuously improved over the years since the 50th. The fatigue life of glass fibre reinforced plastics (GFRP) used in aircraft industry is 10 to 100 times higher compared to glass fibre non crimp fabrics used for wind turbine blades.
To assure a constant and reliable high quality and strength of reinforcement fibres, synthetic fibre production is state of the art (CF, GF). There is a need for recycling GFRP and CFRP waste due to the upcoming use. Pyrolysis and solvolysis are more expensive than the mechanical route however enable a more sustainable recycling. Natural fibres and recycled synthetic fibres have a high scatter in quality and strength. Hence it is a challenge to optimize the production / recycling processes to get a reliable quality for any demanding (second life) application.
Chemical routes for using renewables resources and recycling, is going to be a good approach especially for polymer-matrix systems to get 100% quality (back) compared to the state of the art.
Finally, a proper design, life-time extension and repair is preferable to recycling to keep the carbon footprint as low as possible.
Cryomyces antarcticus, a melanized cryptoendolithic fungus endemic to Antarctica, can tolerate environmental conditions as severe as those in space. Particularly, its ability to withstand ionizing radiation has been attributed to the presence of thick and highly melanized cell walls, which—according to a previous investigation—may contain both 1,8‐dihydroxynaphthalene (DHN) and L‐3,4 dihydroxyphenylalanine (L‐DOPA) melanin. The genes putatively involved in the synthesis of DHN melanin were identified in the genome of C. antarcticus. Most important is capks1 encoding a non‐reducing polyketide synthase (PKS) and being the ortholog of the functionally characterized kppks1 from the rock‐inhabiting fungus Knufia petricola. The co‐expression of CaPKS1 or KpPKS1 with a 4′‐phosphopantetheinyl transferase in Saccharomyces cerevisiae resulted in the formation of a yellowish pigment, suggesting that CaPKS1 is the enzyme providing the precursor for DHN melanin. To dissect the composition and function of the melanin layer in the outer cell wall of C. antarcticus, non‐melanized mutants were generated by CRISPR/Cas9‐mediated genome editing. Notwithstanding its slow growth (up to months), three independent non‐melanized Δcapks1 mutants were obtained. The mutants exhibited growth similar to the wild type and a light pinkish pigmentation, which is presumably due to carotenoids. Interestingly, visible light had an adverse effect on growth of both melanized wild‐type and non‐melanized Δcapks1 strains. Further evidence that light can pass the melanized cell walls derives from a mutant expressing a H2B‐GFP fusion protein, which can be detected by fluorescence microscopy. In conclusion, the study reports on the first genetic manipulation of C. antarcticus, resulting in non‐melanized mutants and demonstrating that the melanin is rather of the DHN type. These mutants will allow to elucidate the relevance of melanization for surviving extreme conditions found in the natural habitat as well as in space.
Aschen und Schlacken aus der thermischen Abfallbehandlung stellen mit rund 25% Massenanteil die größte Reststofffraktion dar. In Deutschland fallen pro Jahr rund 6 Millionen Tonnen an. Während sich die Verwertung der metallischen Anteile problemlos gestaltet, werden an die mineralische Fraktion Anforderungen bezüglich der möglichen Freisetzung von Schwermetallen sowie Chlorid und Sulfat gestellt. In der Ersatzbaustoffverordnung sind für Aschen und Schlacken aus der Abfallverbrennung zwei Materialklassen mit unterschiedlich hohen Anforderungen an die Umweltverträglichkeit definiert. Im Rahmen eines Forschungsprojekts wurde untersucht, ob mit Abtrennung der Feinfraktion von 0–2mm die höhere Materialklasse HMVA-1 erreicht werden kann. Im Ergebnis wurde eine Verbesserung des Auslaugverhaltens erzielt, doch nicht in dem Maß, dass eine Qualität nach HMVA-1 erreicht wird. Rostaschen halten aber die Anforderungen zur Deponierung auf einer DK I-Deponie sowie die Anforderungen zur Verwertung gemäß HMVA-2 sicher ein.
The Great Pacific Garbage Patch, a significant collection of plastic introduced by human activities, provides an ideal environment to study bacterial lifestyles on plastic substrates. We proposed that bacteria colonizing the floating plastic debris would develop strategies to deal with the ultravioletexposed substrate, such as the production of antioxidant pigments. We observed a variety of pigmentation in 67 strains that were directly cultivated from plastic pieces sampled from the Garbage Patch. The genomic analysis of four representative strains, each distinct in taxonomy, revealed multiple pathways for carotenoid production. These pathways include those that produce
less common carotenoids and a cluster of photosynthetic genes. This
cluster appears to originate from a potentially new species of the Rhodobacteraceae family. This represents the first report of an aerobic anoxygenic photoheterotrophic bacterium from plastic biofilms. Spectral analysis showed that the bacteria actively produce carotenoids, such as betacarotene and beta-cryptoxanthin, and bacteriochlorophyll a. Furthermore, we discovered that the genetic ability to synthesize carotenoids is more common in plastic biofilms than in the surrounding water communities. Our findings suggest that plastic biofilms could be an overlooked source of bacteria-produced carotenoids, including rare forms. It also suggests that photoreactive molecules might play a crucial role in bacterial biofilm communities in surface water.
Obwohl der Leichtbau mit carbonfaserverstärkten Kunststoffen (CFK) einen wichtigen Beitrag zum Klimaschutz und zur Ressourceneffizienz liefert, stellt der wachsende Abfallstrom von aktuell 62 kt/a eine große Herausforderung dar. Weil die Produktion von Carbonfasern (CF) überwiegend auf fossilen Rohstoffen basiert und sehr energieintensiv ist, werden nachhaltige Recyclinglösungen dringend benötigt. Hier wird ein Überblick über aktuelle Recyclingprozesse gegeben und deren Limitierungen diskutiert. Darüber hinaus wird eine Möglichkeit zur sicheren chemischen Nutzung von CF-haltigen Reststoffen aufgezeigt, die nicht zur Wiederverwertung geeignet sind. Das Projekt CF Pyro untersucht deren Einsatz als Sekundärrohstoff in der Pyrometallurgie und setzt besondere Schwerpunkte bei der Reaktivität von CF, der Prozessstabilität und der Vermeidung von Emissionen gesundheitsschädlicher WHO-Fasern. Abschließend wird eine, auf den experimentellen Ergebnissen und im Dialog mit Expertinnen und Experten aus Wirtschaft, Wissenschaft und Politik entwickelten, Technologiebewertung vorgestellt.
Obwohl der Leichtbau mit carbonfaserverstärkten Kunststoffen (CFK) einen wichtigen Beitrag zum Klimaschutz und zur Ressourceneffizienz liefert, stellt der wachsende Abfallstrom von aktuell 62 kt/a eine große Herausforderung dar. Weil die Produktion von Carbonfasern (CF) überwiegend auf fossilen Rohstoffen basiert und sehr energieintensiv ist, werden nachhaltige Recyclinglösungen dringend benötigt. Hier geben wir einen Überblick über aktuelle Recyclingprozesse und diskutieren deren Limitierungen. Darüber hinaus zeigen wir eine Möglichkeit zur sicheren chemischen Nutzung von CF-haltigen Reststoffen auf, die nicht zur Wiederverwertung geeignet sind. Das Projekt CF Pyro untersucht deren Einsatz als Sekundärrohstoff in der Pyrometallurgie und setzt besondere Schwerpunkte bei der Reaktivität von CF, der Prozessstabilität und der Vermeidung von Emissionen gesundheitsschädlicher WHO-Fasern. Nach der Präsentation der Projektergebnisse erfolgte in der Diskussion mit Expertinnen und Experten aus Wirtschaft, Wissenschaft und Politik eine Technologiebewertung für die Nutzung von CF in pyrometallurgischen Prozessen.