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Eingeladener Vortrag
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A wide variety of fungi and bacteria are known to contaminate fuels and fuel systems. These microbial contaminants have been linked to fuel system fouling and corrosion. The fungus Hormoconis resinae, a common jet fuel contaminant, is used in this study as a model for developing innovative risk assessment methods. A novel qPCR protocol to detect and quantify H. resinae in, and together with, total fungal contamination of fuel systems is reported. Two primer sets, targeting the markers RPB2 and ITS, were selected for their remarkable specificity and sensitivity. These primers were successfully applied on fungal cultures and diesel samples demonstrating the validity and reliability of the established qPCR protocol. This novel tool allows clarification of the current role of H. resinae in fuel contamination cases, as well as providing a technique to detect fungal outbreaks in fuel systems. This tool can be expanded to other well-known fuel-deteriorating microorganisms.
Sub-aerial biofilms (SAB) are ubiquitous, self-sufficient microbial ecosystems found on mineral surfaces at all altitudes and latitudes. SABs, which are the principal causes of weathering on exposed terrestrial surfaces, are characterized by patchy growth dominated by associations of algae, cyanobacteria, fungi and heterotrophic bacteria. A recently developed in vitro system to study colonization of rocks exposed to air included two key SAB participants - the rock-inhabiting ascomycete Knufia petricola (CBS 123872) and the phototrophic cyanobacterium Nostoc punctiforme ATCC29133. Both partners are genetically tractable and we used them here to study weathering of granite, K-feldspar and plagioclase. Small fragments of the various rocks or minerals (1–6 mm) were packed into flow-through columns and incubated with 0.1% glucose and 10 μM thiamine-hydrochloride (90 μL min−1) to compare weathering with and without biofilms. Dissolution of the minerals was followed by: (i) analysing the degradation products in the effluent from the columns via Inductively Coupled Plasma Spectroscopy and (ii) by studying polished sections of the incubated mineral fragments/grains using scanning electron microscopy, transmission electron microscopy and energy dispersive X-ray analyses. K. petricola/N. punctiforme stimulated release of Ca, Na, Mg and Mn. Analyses of the polished sections confirmed depletion of Ca, Na and K near the surface of the fragments. The abrupt decrease in Ca concentration observed in peripheral areas of plagioclase fragments favored a dissolution-reprecipitation mechanism. Percolation columns in combination with a model biofilm can thus be used to study weathering in closed systems. Columns can easily be filled with different minerals and biofilms, the effluent as well as grains can be collected after long-term exposure under axenic conditions and easily analyzed.
The interface between materials and the environment is populated by microorganisms which organize on surfaces to form specially adapted and resistant microbial associations, so-called biofilms. Surface-bound biofilm growth is associated with substantial secretion of metabolic products that can significantly influence material corrosion. Here, a group of material-inhabiting fungi are presented, which colonize and weather air-exposed materials such as building façades, roofs and solar systems. A selected model fungus is used to carry out genetic and molecular biology experiments, which are microbiologically cutting-edge and allow for the development of novel time-saving techniques for material testing. The degree of material damage under the influence of this model fungus shows a measure of the aggressiveness of the biofilm under defined environmental conditions - and thus explores fundamental components of the interaction between material and biofilm.
The interface between materials and the environment is populated by microorganisms which organize on surfaces to form specially adapted and resistant microbial associations, so-called biofilms. Surface-bound biofilm growth is associated with substantial secretion of metabolic products that can significantly influence material corrosion. Here, a group of material-inhabiting fungi are presented, which colonize and weather air-exposed materials such as building façades, roofs and solar systems. A selected model fungus is used to carry out genetic and molecular biology experiments, which are microbiologically cutting-edge and allow for the development of novel time-saving techniques for material testing. The degree of material damage under the influence of this model fungus shows a measure of the aggressiveness of the biofilm under defined environmental conditions - and thus explores fundamental components of the interaction between material and biofilm.
Biofilm formation on materials leads to high costs in industrial processes, as well as in medical applications. This fact has stimulated interest in the development of new materials with improved surfaces to reduce bacterial colonization. Standardized tests relying on statistical evidence are indispensable to evaluate the quality and safety of these new materials. We describe here a flow chamber system for biofilm cultivation under controlled conditions with a total capacity for testing up to 32 samples in parallel. In order to quantify the surface colonization, bacterial cells were DAPI (4‘,6-diamidino-2-phenylindole)-stained and examined with epifluorescence microscopy. More than 100 images of each sample were automatically taken and the surface coverage was estimated using the free open source software g’mic, followed by a precise statistical evaluation. Overview images of all gathered pictures were generated to dissect the colonization characteristics of the selected model organism Escherichia coli W3310 on different materials (glass and implant steel). With our approach, differences in bacterial colonization on different materials can be quantified in a statistically validated manner. This reliable test procedure will support the design of improved materials for medical, industrial, and environmental (subaquatic or subaerial) applications.
Genetic manipulation of protective pigments in a rock-inhabiting model fungus Knufia petricola A95
(2016)
Sub-aerial biofilms typically form on bare rock. They consist of 99% cell material and extracellular polymeric substances (EPS) metabolising at low water availability. Rock-inhabiting melanised fungi represent an important part of the microbial community in these environments, playing important roles in the colonisation of mineral surfaces, rock weathering and soil formation in the ecological/geochemical context. Different cellular stress responses make rock-inhabiting ascomycetes fit for survival under extremely changing irradiation, as well as water, energy sources and nutrient availability. Melanised, rock-inhabiting fungi possess multiple protective pigments, form facultative symbiotic associations with photobionts and weather minerals. Melanised fungi build a protective layer around the cell that is critical in adhesion to other living partners, for the colonisation of the substrate and in the subsequent damage of the colonised surface. We chose Knufia petricola (Chaetothyriales) as a model species to analyse colonisation of surfaces. The basic physiology of K. petricola strain A95 is studied, its full genome sequence has been prepared for annotation and methods for deleting specific genes have been established. Unique features of K. petricola including the protective pigments (melanin and carotenoids) and EPS/cell wall properties are now being dissected genetically. As K. petricola strain A95 is in the basic clade of Chaetothyriales, it is an ancestor of both important human pathogens including Exophiala and lichens from the Verrucariaceae family. For this reason studies with A95 can help clarify the basis of fungal pathogenicity – as well as explain interactions with microscopic phototrophic partners like unicellular green algae and cyanobacteria. With Knufia petricola we will establish a canon of experimental approaches to characterise and quantify fungi that actively contact inanimate solid materials. The set of methods developed for Knufia will be adapted to heavily melanised and EPS-producing ascomycetes and can be broadly applied to medically important as well as material-colonising fungi.
A new tool in material science - Targeted disruption of melanin synthesis in rock-inhabiting fungi
(2016)
The ability to survive almost absolute dehydration through air-drying is a remarkable feature of rock-inhabiting microcolonial fungi (MCF), which colonise rock surfaces in hot and cold deserts. Understanding of the underlying mechanisms which allow this group of fungi to conquer natural and man-made environments requires a set of modern biological techniques and approaches that are under development in our laboratory. We will present an overview of the targeted disruption of melanin biosynthesis genes in the rock-inhabiting Knufia petricola and give inside into the lines of research and the network of supporting laboratories that made this progress possible.
Fungal extracellular materials reinforce a constant interaction between their cell wall and the environment. A dynamic mixture of chitin, glucans, mannans, glycoproteins, glycolipids and pigments supports the success of all fungal life styles – from symbiotic to the free-living and pathogenic. Fungi are perfectly adapted to grow on surfaces and in porous environments, where they form medically and geochemically relevant biofilms. Fungal EPS are critical in adhesion to other fungi, other cells or substratum as well as in the following interaction with the host immune system or material they attack, degrade and deteriorate respectively. Characterisation of extracellular compounds and understanding of its function is necessary to limit damage caused by fungal activity. All necessary methodology from chemical characterization to complete genetic analyses has been developed for medically important fungi. Now it is time to apply this knowledge to the numerous, largely aerobic and very active organisms that occupy a wide range of atmosphere-exposed habitats in the upper lithosphere. One can expect that analogies between medically- and environmentally-relevant model fungal species will help us to address the dynamics of the fungal cell EPS matrix in much more efficient and widely applicable ways.
Essential processes in the establishment and maintenance of rock biofilms include photosynthesis, production of extracellular polymeric substances, substrate penetration and atmospheric nutrient enrichment. Functional diversity is supported by a complex subaerial biofilm (SAB) community of heterotrophic and phototrophic microorganisms. Stress tolerant and melanised Ascomycetes dominate heterotrophic SABs while diverse algae and cyanobacteria comprise the phototrophic consortia. Laboratory simulation of SABs permits use of molecular-genetic methods coupled with geochemical and microscopic analyses to study weathering. Our in vitro model includes two free-living and symbiosis-competent, genetically tractable microorganisms: the cyanobacterium Nostoc punctiforme and the microcolonial rock-inhabiting fungus Knufia petricola. This genetically amenable cyanobacterium/fungus model biofilm allows development of quantitative methods tailored to the natural diversity of SABs. Precise, reproducible studies using this model biofilm have shown that both the melanised fungus, as well as the combined bacterial/fungal system, enhances the weathering of minerals. Geochemical signatures of these in vitro rock biofilms can now be obtained and compared with bacterial/fungal mutants of varied EPS composition and substrate penetration patterns. And finally, precise study of the model cyanobacterium/fungus biofilm will Permit prediction of the effects of conservational treatments.
A biological impact on weathering was recognized already at the beginning of the twentieth century, when biochemical influence of the lichen growth on rocks was convincingly demonstrated. Later it was shown that the
progress of solid rock weathering initiated by biological colonization was affected by the initial porosity system and sensitivity of mineral association. In the meantime
a considerable amount of diverse scientific data confirm the importance of biological rock colonizers (lichens and free-living rock biofilms) in mineral material dynamics as they occur at the atmosphere-exposed rock surfaces on local as well as global scale. Subaerial rock biofilms—microbial ecosystem including free-living heterotrophic and phototrophic settlers of bare rock surfaces—are characteristic for the first stage of primary succession of terrestrial ecosystems on mineral substrates.
These cultivable and free-living communities are dominated by fungi and set the stage for the later development of a lichen cover, but in comparison to lichens also represent a new tool for laboratory experimentation and thus open a new stage of work in geomicrobiology. The Minerals sensitivity to microbially induced biological weathering can be demonstrated by studies of natural samples as well as by the laboratory mesocosm experiments.