4 Material und Umwelt
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Over the past two decades, per- and polyfluoroalkyl substances (PFASs) have emerged as worldwide environmental contaminants, calling out for sophisticated treatment, decomposition and remediation strategies. In order to mineralize PFAS pollutants, the incineration of contaminated material is a state-of-the-art process, but more cost-effective and sustainable technologies are inevitable for the future. Within this review, various methods for the reductive defluorination of PFASs were inspected. In addition to this, the role of mechanochemistry is highlighted with regard to its major potential in reductive defluorination reactions and degradation of pollutants. In order to get a comprehensive understanding of the involved reactions, their mechanistic pathways are pointed out. Comparisons between existing PFAS decomposition reactions and reductive approaches are discussed in detail, regarding their applicability in possible remediation processes. This article provides a solid overview of the most recent research methods and offers guidelines for future research directions.
Biofilms cause complications and high costs in both industry and medicine. Of particular interest are bacterial infections of prosthetic materials, which usually cannot be eliminated due to the high antibiotic resistance known for bacteria forming biofilms. The search for new materials and coatings with lower colonization potential and antibacterial activity is of great importance to reduce biofilm formation. However, there is no standardized procedure to examine the colonization characteristics of bacteria in the Biofilm state in situ. Here, we describe an automated epifluorescence microscopy system for the semi-quantitative analysis of three-dimensional (3D) biofilms on various surfaces. To analyze adherent bacteria, three materials (glass, steel and titanium) were incubated with bacteria in a flow chamber system. After fluorescence staining of the bacteria, automated image capturing, quantification of the bacteria, measurement of the colonized area and determination of the 3D biofilm height were carried out by using novel software.
Furthermore, the materials were examined for their surface topography using white light scanning interferometry. Titanium compared to glass showed a significantly higher number of adherent bacteria.
We argue that this was due to the higher microroughness of titanium. The colonized area was in accordance with the number of adherent bacteria and was also significantly larger on titanium coupons compared to glass. Maximum 3D biofilm height on glass coupons was significantly lower compared to the ones on steel and titanium. This novel method enables the standardized, automated investigation of the colonization with bacteria on different materials. This approach can considerably support the characterization of new material surfaces and their innovative coatings by analyzing the amount of attached Bacteria and thickness of biofilms in situ and eliminates the need of conventional cultivation.
Biocides are in contact with soils through direct application and passive leaching from protected materials. Consequently, soil microorganisms are exposed to biocides even though they are not their primary targets. Soil is a large reservoir of microbial diversity and has been hypothesized to be a crucial factor for the evolution and spread of antimicrobial resistance.
Currently, there is little knowledge on how biocides used to protect materials affect the evolution and spread of resistance. Thus, our aim is to investigate the risk for the evolution of biocide resistance and cross-resistance to antibiotics. In addition, we aim to elucidate the affect of biocides on the spread of resistance via horizontal gene transfer (HGT).
In adaptive laboratory evolution experiments we cultured selected model soil microorganism with representative biocides under selection regimes with increasing and stable biocide concentrations followed by antibiotic and biocide cross-resistance determination. Moreover, we investigate if the selected biocides affect the rates of de novo mutations and HGT of plasmids that carry resistance genes among soil microorganism.
Our results show only small increases of biocide resistance during serial transfers under increasing biocide concentrations. One reason for this might be the narrow selective window for biocide resistance due to steep dose-response relationships. Furthermore, our results indicate that a stable low-level biocide regime did not select for high level cross-resistance to antibiotics and other biocides.
Moreover, material preservatives affected the rates of HGT via conjugation and the mutation rates at sub-inhibitory concentrations. The results will enable future risk assessment regarding resistance evolution for biocides used as material preservatives.
The suitability of common analytical methods for the determination of active substances from wood preservatives in aged wood samples was investigated during an interlaboratory study. Permethrin, propiconazole and tebuconazole were quantified in 1.5 and 8 year-old wood samples by gas chromatography and liquid chromatography. Generally, the applied Methods yielded reliable results for these samples. However, wood components can coelute with propiconazole and tebuconazole during liquid chromatography. Optimization of separation might be required if UV detection is applied.
Terrestrial mud volcanoes are unique structures driven by tectonic pressure and fluids from the deep subsurface. These structures are mainly found in active tectonic zones, such as the area near the Los Bajos Fault in Trinidad. Here we report a chemical and microbiological characterization of three mud volcanoes, which included analyses of multiple liquid and solid samples from the mud volcanoes. Our study confirms previous suggestions that at least some of the mud volcano fluids are a mixture of deeper salt-rich water and surficial/precipitation water. No apparent water quality differences were found between sampling sites north and south of a major geological fault line. Microbiological analyses revealed diverse communities, both aerobic and anaerobic, including sulfate reducers, methanogens, carbon dioxide fixing and denitrifying bacteria. Several identified species were halophilic and likely derived from the deeper salt-rich subsurface water, while we also cultivated pathogenic species from the Vibrionaceae, Enterobacteriaceae, Shewanellaceae, and Clostridiaceae. These microorganisms were likely introduced into the mud volcano fluids both from surface water or shallow ground-water, and perhaps to a more minor degree by rain water. The identified pathogens are a major health concern that needs to be addressed.
Dihydroxynaphthalene (DHN) melanin is produced by different Ascomycetes via slightly differing biosynthetic routes. The polyketide synthases (PKS) release the heptaketide YWA1, the hexaketide AT4HN or the pentaketide T4HN. The first two products are deacetylated by ‘yellowish-green’ hydrolases to T4HN, and T4HN is further converted by a core set of enzymes to DHN. Final polymerization steps are accomplished by multicopper oxidases. DHN melanogenesis is often regulated in a spatial and temporal fashion resulting e.g. in melanized reproduction, survival and/or infection structures. Remarkable is the regulation of the DHN melanogenesis in the foliar plant pathogen Botrytis cinerea: it involves two differently expressed PKSs providing the precursor in conidia and sclerotia, respectively (Schumacher 2016, Mol Microbiol). In contrast, a polyphyletic group of Ascomycetes (microcolonial fungi/ black yeast) dwelling in hostile habitats such as bare rock surfaces in hot and cold deserts, exhibits constitutive melanogenesis. Here, DHN melanin builds a protective layer around all vegetative cells thus contributing to the survival of diverse environmental stresses even without specialized reproduction structures. As part of our continuing research on microcolonial rock-inhabiting fungi, we chose the genetically amenable Knufia petricola strain A95 (Nai et al. 2013, Fungal Genet Biol; Noack-Schönmann et al. 2014, AMB Express) for detailed studies. DHN-deficient mutants generated by targeted mutation of biosynthetic genes were studied with regard to the architecture of the cell wall and the EPS (extracellular polymeric substances) matrix, attachment to and weathering of olivine, as well as the tolerance to abiotic and biotic stresses. We will discuss the critical role of the outer cell surface (DHN melanin and EPS) in adhesion to the substrate and subsequent damage of the colonized surface.
Microcolonial black fungi, a polyphyletic group of ascomycetes, exhibit constitutive melanin formation, yeast-like growth and high stress tolerances. They dominate – often together with bacteria and algae in sub-aerial biofilms – a range of hostile environments including natural and man-made ones, from salterns to dishwashers, roofs and solar panels. Because of lacking genetic tools and the slow growth of most isolates, the genetic bases for these specific properties are largely unknown. The rock-inhabiting fungus Knufia petricola (Eurotiomycetes, Chaetothyriales) exhibits all characteristics of microcolonial black fungi and was selected as recipient for genetic engineering to study gene functions and genetic interactions. Different variants of green and red fluorescent proteins were successfully expressed indicating that fluorescence microscopy using genetically encoded fluorescent proteins and fluorescent dyes enables various cell biology approaches. Furthermore, genes of biosynthetic pathways (DHN melanin, carotenoids, uracil, adenine) were successfully mutated by applying traditional gene replacement and plasmid-based or ribonucleoprotein (RNP)-based CRISPR/Cas9 or silenced by RNA interference (RNAi). The availability of this advanced and efficient genetic toolbox and the annotated genome sequence of strain A95 makes K. petricola an excellent model for exploring the secrets of microcolonial black fungi.
Fungi that share light-flooded habitats with phototrophs may profit from their excess photosynthetic products. But to cope with sunlight-associated stresses [e.g. high temperatures, UV radiation with associated DNA damage, accumulation of reactive oxygen species (ROS), desiccation and osmotic stresses] it is important for fungi to accurately sense and respond to changes in light. To test the hypothesis that light is an environmental cue that Ascomycota use to coordinate growth, stress responses as well as to establish pathogenic or symbiotic relationships, the photoreceptor (PR) distribution in species from different ecological niches was analysed. The genomes of black [dihydroxynaphthalene (DHN) melanin-containing] fungi from phyllosphere and exposed solid surfaces contain multiple photoreceptors (PRs). The plant pathogen Botrytis cinerea (Leotiomycetes) has a highly sophisticated photosensory and signalling system that helps to avoid light and to locate susceptible hosts. Rock-inhabiting Dothideomycetes and Eurotiomycetes including Knufia petricola possess equal numbers ofPRs along with the same set of protective pigments. This similarity between black fungi from plant and rock surfaces suggests that photoperception and -regulation are important for fungi that receive nutrients through cooperation with phototrophs. Genetic tools for manipulating K. petricola exist and will be used to test this idea.
The polyphyletic group of black fungi within the Ascomycota (Arthoniomycetes, Dothideomycetes, and Eurotiomycetes) is ubiquitous in natural and anthropogenic habitats. Partly because of their dark, melanin-based pigmentation, black fungi are resistant to stresses including UV- and ionizing-radiation, heat and desiccation, toxic metals, and organic pollutants. Consequently, they are amongst the most stunning extremophiles and poly-extreme-tolerant organisms on Earth. Even though ca. 60 black fungal genomes have been sequenced to date, [mostly in the family Herpotrichiellaceae (Eurotiomycetes)], the class Dothideomycetes that hosts the largest majority of extremophiles has only been sparsely sampled. By sequencing up to 92 species that will become reference genomes, the “Shed light in The daRk lineagES of the fungal tree of life” (STRES) project will cover a broad collection of black fungal diversity spread throughout the Fungal Tree of Life. Interestingly, the STRES project will focus on mostly unsampled genera that display different ecologies and life-styles (e.g., ant- and lichen-associated fungi, rock-inhabiting fungi, etc.). With a resequencing strategy of 10- to 15-fold depth coverage of up to ~550 strains, numerous new reference genomes will be established. To identify metabolites and functional processes, these new genomic resources will be enriched with metabolomics analyses coupled with transcriptomics experiments on selected species under various stress conditions (salinity, dryness, UV radiation, oligotrophy). The data acquired will serve as a reference and foundation for establishing an encyclopedic database for fungal metagenomics as well as the biology, evolution, and ecology of the fungi in extreme environments.