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Biochemische und physiologische Charakterisierung der extrazellulären Matrix eines Modellbiofilms
(2018)
An der Grenzschicht zwischen einer festen Oberfläche und der umgebenden Luft wachsende subaerische Biofilme (SAB) zeichnen sich durch eine erhöhte Toleranz gegenüber extremen Umweltbedingungen und dem Eintrag von Bioziden aus. Dieser Schutz vor äußeren Umwelteinflüssen wird vornehmlich durch den Beitrag von extrazellulären polymeren Substanzen (EPS) und Pigmenten, wie Melanin und Carotinoiden, vieler unterschiedlicher Organismen gewährleistet. Deren Synthese wird wiederum durch intrazelluläre Botenstoffe reguliert. Das Cyanobakterium Nostoc punctiforme und der mikrokoloniale Pilz Knufia petricola, als Partner eines etablierten SAB-Modells und Vertreter zweier typischer Organismengruppen in SAB, wurden genutzt, um Botenstoffe und Pigmente genetisch zu manipulieren und die Biofilmmatrix dieser Mutanten strukturell zu untersuchen. In dieser Arbeit konnten erstmals die EPS beider Organismen extrahiert und die Struktur der extrazellulären Polysaccharide beschrieben werden. Daneben wurden die extrazellulären Polysaccharide von K. petricola Wildtyp mit denen verschiedener Pigmentmutanten verglichen. Das Fehlen des Schutzpigmentes Melanin führte zu einer ausgeprägteren extrazellulären Matrix in den Biofilmen. Gleichzeitig änderte sich die Struktur der extrazellulären Polysaccharide. Während der Wildtyp zu ~80% ein α-Glucan und zu ~20% ein α/β-Galaktomannan sekretierte, war der Anteil des Galaktomannans bei den Melaninmutanten erhöht. Das Ausschalten der Carotinoid-Synthese hatte jedoch keinen Einfluss auf die Beschaffenheit der extrazellulären Polysaccharide. Ein deutlich komplexeres extrazelluläres Polysaccharid aus acht verschiedenen Monosaccharid-Einheiten bildete N. punctiforme. Durch die Überproduktion des bakteriellen sekundären Botenstoffes c-di-GMP konnte zudem ein grundlegender Einfluss auf die Reaktion des Cyanobakteriums gegenüber externen Signalen und die damit verbundene Zelldifferenzierung gezeigt werden. Ein artifiziell erhöhtes c-di-GMP-Level in den Zellen führte zur Ausbildung eines sessilen Lebensstils durch Hemmung der Differenzierung motiler Hormogonien und vermehrte Produktion von EPS. Die Struktur der extrazellulären Polysaccharide wurde dadurch nicht verändert. Neben der strukturellen Analyse konnten die Veränderungen der extrazellulären Matrix beider Organismen zusätzlich durch mikroskopische Methoden visualisiert werden. Beide Organismen steuern komplexe Polymere, deren Produktion maßgeblich mit intrazellulären Faktoren verknüpft ist, zu der extrazellulären Matrix des Modellbiofilms bei.
Melanised cell walls and extracellular polymeric matrices protect rock-inhabiting microcolonial fungi from hostile environmental conditions. How extracellular polymeric substances (EPS) perform this protective role was investigated by following development of the model microcolonial black fungus Knufia petricola A95 grown as a sub-aerial biofilm. Extracellular substances were extracted with NaOH/formaldehyde and the structures of two excreted polymers studied by methylation as well as NMR analyses. The main polysaccharide (~ 80%) was pullulan, also known as α-1,4-; α-1,6-glucan, with different degrees of polymerisation. Αlpha-(1,4)-linked-Glcp and α-(1,6)-linked-Glcp were present in the molar ratios of 2:1. A branched galactofuromannan with an α-(1,2)-linked Manp main chain and a β-(1,6)-linked Galf side chain formed a minor fraction (~ 20%). To further understand the roles of EPS in the weathering of minerals and rocks, viscosity along with corrosive properties were studied using atomic force microscopy (AFM). The kinetic viscosity of extracellular K. petricola A95 polysaccharides (≈ 0.97 × 10-6 m2 s-1) ranged from the equivalent of 2% (w/v) to 5% glycerine, and could thus profoundly affect diffusion-dominated processes. The corrosive nature of rock-inhabiting fungal EPS was also demonstrated by its effects on the aluminium coating of the AFM cantilever and the silicon layer below.
Black ascomycetous microcolonial fungi (MCF) are persistent inhabitants of rock surfaces in deserts as well as ubiquitous in other wide-spread terrestrial ecosystems including man-made materials such as solar panels. The ability of MCF to cope with multiple, rapidly fluctuating stresses makes the group an interesting subject in the study of stress resistance. Carotenoids and melanin are amongst the protective pigments that contribute to the robustness of MCF. We studied their role in stress resistance of the non-pathogenic rock-inhabiting fungus Knufia petricola (Chaetothyriales) strain A95. Disruption of melanin synthesis affects not only oxidative-stress resistance but also the extracellular polysaccharide (EPS) matrix and lipid composition of the cell membrane.
The role of c-di-GMP in biofilm formation of the filamentous cyanobacterium Nostoc punctiforme
(2016)
The signaling molecule c-di-GMP is a ubiquitous second messenger in bacterial species including cyanobacteria. C-di-GMP stimulates the biosynthesis of extracellular polymeric substances (EPS) in biofilms and regulates the transition between a motile and a sessile life style in many bacteria. In our study we focus on c-di-GMP function and EPS production associated with biofilm Formation in Nostoc punctiforme, which is known to produce EPS in biofilms and liquid culture.
Genetic manipulation of protective pigments in a rock-inhabiting model fungus Knufia petricola A95
(2016)
Black ascomycetous microcolonial fungi are persistent inhabitants of rock surfaces, but are especially conspicuous in hostile environments like cold and hot deserts. The unique robustness of MFCs is supported by protective pigments, like melanin and carotenoids. We study the role of these pigments in the stress resistance of the model rock fungus Knufia petricola (Chaetothyriales) strain A95.
Several fires involving ETIC (external thermal insulation composite) systems with polystyrene foam insulation in Germany led to an extensive discussion about fire safety of such systems. A collection initiated by the Frankfurt fire service of façade fires which include polystyrene insulation foam shows that especially fires which started in front of the buildings led to more severe fires of the façade than fires that started inside the buildings. In several fires the ignition source were burning waste containers. Three large scale tests which were initiated by German building ministries showed weaknesses of the existing systems when challenged by a bigger fire source in front of the façade. Since then measures have been introduced to enhance these systems and an additional test with a 200 kg wood crib in front of a large test rig has been used for approval of ETICS. However, the recently introduced German draft standard DIN E 4102-20 does not take these changes into account although real cases and the large scale tests showed that fire scenarios with a bigger ignition source as a waste container are not covered by the DIN E 4102-20. Numerical investigations show that regarding the heat flux to the area above the opening (e.g. a window) also only a fraction of real fires is covered. Additionally damaged systems have been investigated using the Single Burning Item (SBI) test with higher heat release rates of the burner. The damage significantly influenced the fire development of the specimen. Collapse of a damaged coating of an ETIC system occurred during the test and had a sudden fire growth as a result as the whole specimen was suddenly on fire. Several effects which could be seen in the intermediate scale tests correspond to observations which were made in the real cases. In Germany insulation of existing buildings is often enhanced with application of ETICS. In most cases the buildings are in use at the time when the construction takes place. At several stages of the construction process large amounts of unprotected polystyrene are stored in immediate proximity of the building and unprotected polystyrene can be in place on the façade for several weeks. As a consequence of the investigations challenges and possible measures to enhance fire safety of ETIC systems are discussed.
The new standard ISO 176362:2013 'NDT of welds: Radiographic testing - Part 2: X- and gamma ray techniques with digital detectors' describes a complex procedure for film replacement by phosphor imaging plates and digital detector arrays. RT modeling software should consider these detector types, X-ray film, and the standard requirements for image quality. Practitioners expect the same visibility of image quality indicators (IQI) in the simulated radiographs as in the experimental exposures. The proposed benchmark test is based on the comparison of experimental radiographs taken at BAM with modeled ones of participants. The experimental setup and the determination of the equivalent penetrameter sensitivity (EPS) as described in the procedure of ASTM E 746 are used for quantitative evaluation of the achievable contrast sensitivity for step hole IQIs as considered in Annex B of ISO 17636-2. System classification data for Computed Radiography (CR) and film systems will be provided by BAM according to ISO 116991 for selected film systems and according to ASTM E 2446 for selected CR systems. The classification of films and digital detectors is based on the measurement of the dose response function, the basic spatial resolution (SRb) of the image, and the measured image noise, which depends on the detector efficiency, the quantum statistics, and the detector fixed pattern noise.