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DHN (1,8-dihydroxynaphthalene) 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 (MCOs). The involved genes are tightly clustered, partially clustered or widely distributed in the genomes of DHN melanin-producing fungi. DHN melanogenesis is often regulated in a spatial and temporal fashion resulting e.g. in melanized reproduction, survival and/or infection structures. 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, we report on the identification and functional characterization of the DHN melanogenic genes of Knufia petricola as a representative of the Chaetothyriales, the sister order of the Eurotiales. Orthologs for all melanogenic genes were identified in the genome of K. petricola A95, including one gene encoding the polyketide synthase (KpPKS1), two genes encoding ‘yellowish-green’ hydrolases (KpYGH1,2), two genes encoding THN reductases (KpTHR1,2) and one gene encoding a scytalone dehydratase (KpSDH1). Ten genes encoding MCOs were identified, all MCOs are predicted to be secreted. The genes are not clustered in the genome but are highly expressed. Gene functions are studied by generation of single, double, and multiple deletion mutants in K. petricola and by heterologous expression in Saccharomyces cerevisiae for reconstruction of the synthesis pathway.
DHN (1,8-dihydroxynaphthalene) melanin is produced by Ascomycetes via slightly differing synthetic routes. Polyketide synthases release YWA1, AT4HN or T4HN. YWA1 and AT4HN are deacetylated by ‘yellowish-green’ hydrolases, and T4HN is converted by a core set of enzymes to DHN. Final polymerization steps are accomplished by multicopper oxidases. The melanogenic genes are tightly, partially or not clustered in the genomes, and are often regulated in a spatial and/or temporal fashion. By contrast, microcolonial fungi/black yeasts – a polyphyletic group of Ascomycetes dwelling in hostile habitats such as bare rock surfaces – feature constitutive DHN melanogenesis. Here, we report on the DHN melanogenic genes of Knufia petricola (Eurotiomycetes/Chaetothyriales).
Dihydroxynaphthalene (DHN) melanin is produced by diverse 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 and survival structures of the foliar plant pathogen Botrytis cinerea (Schumacher 2016, Mol Microbiol). In contrast, microcolonial black fungi exhibit 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. For studying the regulation and relevance of DHN melanogenesis for tolerance of abiotic and biotic stresses, adhesion to substrates and subsequent damage of colonized surfaces, the rock-inhabiting fungus Knufia petricola was chosen as gene functions in this fungus can be studied by CRISPR/Cas9-based genome editing. The putative melanogenic genes were identified in the genome of K. petricola, deleted to confirm their involvement in DHN melanogenesis and co-expressed in Saccharomyces cerevisiae for reconstruction of the synthesis pathway. Phenotypes of deletion mutants are studied for specifying the functions of DHN melanin in K. petricola.
Per- and polyfluoroalkyl substances (PFAS) are a large group of organofluorine surfactants used in the formulations of thousands of consumer goods, including aqueous film-forming foams (AFFF) used to suppress aviation fires in training scenarios, non-stick cookware, fast-food wrappers, water-repellent fabrics, medical equipment, and plastic and leather products. As a result of the perpetual use of PFAS containing products, effluents and sewage sludge from wastewater treatment plants (WWTPs) have been observed to be an important pathway for PFAS into the environment. In Germany, phosphorus and other nutrients from sewage sludge and wastewater should be recycled in WWTPs of cities with a large population. However, it is not clear if PFAS contamination from wastewater and sewage sludge end up in novel wastewater-based fertilizers. Normally, PFAS are analyzed using PFAS protocols typically with time-consuming extraction steps and LC-MS-MS quantification. However, for screening of PFAS contaminations in sewage sludge or wastewater-based fertilizers also passive sampler based on the Diffusive Gradients in Thin-films (DGT) technique can be used for the PFAS extraction. Afterwards, combustion ion chromatography (CIC) can be applied to analyse the “total” amount of PFAS on the passive sampler. Here, we show results from the DGT method in comparison to those of the extractable organic fluorine (EOF) method for a variety of wastewater-based fertilizers. Additionally, we analysed the adsorption of PFAS on the weak anion exchanger (WAX) based DGT passive sampler binding layer by infrared and fluorine K-edge X-ray adsorption near-edge structure (XANES) spectroscopy.
Brazilian farming industry consumed around 2.2 million tons of phosphorus (P) fertilizers in 2016. The agricultural industry depends on imported P fertilizers and over 98% of P fertilizers were used for sugarcane, soybean and maize production.
An alternative is to use P from sugarcane bagasse. Bagasse is the fibrous plant material remaining after extraction of sugarcane juice, and it is combusted for energy production. Remaining ash con-tains up to 0.6 wt% P. The use of bagasse ash (BA) as P fertilizer could decrease the annual import of P fertilizers by 6% of the imported P fertilizer based on 2016 values. Since the bioavailability of P from BA to plants is poorly investigated, this study addresses the effects of (i) gasification tempera-ture (710-849°C), (ii) processing method (gasification vs. combustion), (iii) biomass modifications by co-processing bagasse with chicken manure (BA+CM), and (iv) the soil (Brazilian Oxisol soil vs. nutri-ent poor substrate) on the bioavailability of P from BA to soybeans (Glycine max).
Gasification of BA at 806 °C resulted in significantly highest uptake of P by soybeans and was around 0.33 mmol after 51 days growing. The bioavailability of P significantly increased due to co-gasification of bagasse and chicken manure (BA+CM) and the soybeans took up around 16% more P. Compared to the nutrient-poor substrate, the bioavailability of P in BA+CM ash treated Oxisol soil was signifi-cantly lower by 46% and there was no significant effect of processing method on the bioavailability of P from the BA+CM ash to soybeans. Contrary to the Oxisol soil, the bioavailability of P from co-combusted BA+CM ash was significantly higher compared to co-gasified BA+CM ash. In conclusion, co-processing of bagasse with nutrient rich residues can increase the value of BA as P fertilizer. The bioavailability of P from ash to plants depends on the P forms. Mineralogical analyses of ash P forms by NMR and X-ray diffraction are in progress and will be presented at the conference.
The church of Koszewko (Poland) is a brick building edified in the 15th century built on cobblestone foundations. There are five windows in the sanctuary. Three of them enclose heraldic panels from the Küssow’s family from the 15th century which are surrounded with Goethe glass from the 18th century to complete the windows. The colored heraldic panels are strongly damaged and corroded with massive paint layer losses, glass- and leadbreakages. Those medieval glass fragments have been shortly discovered and are of particular interest for Poland since only few medieval glazing have been conserved.
The damages as well as the glass compositions have been investigated with ESEM/EDX. Two categories of medieval glass compositions have been identified. The blue glass is particularly sensible to corrosion because of his high content in K2O. The colorless and the red glass samples belong to a stable glass type. Due to the thickness of the gel layer, it is easy to see that the degradation is strongly proceeded. The protection of those medieval stained-glass panels is absolute necessary.
The medieval panels have been restored and surrounded from a copper frame. Then they have been fixed on the wood frame in the church. The exterior glazing has been closed with a panel of Goethe glass. The gap between the Goethe- and the medieval glass is about 3 cm. The Goethe glass panel has been stabilized with a film based on polyester to protect the medieval glasses against any damages. In this way, a low cost protective glazing has been installed for a long-term conservation of each medieval stained-glass panels. The climate measurements over the period of one year on the restored windows are in process. The temperature and the relative humidity are recorded in the church interior, in the gap between the original and the Goethe glass and outdoors.
In Vorbereitung einer Restaurierung erfolgten naturwissenschaftliche Untersuchungen zu Schadensphänomenen und Glaszusammensetzungen an mittelalterlichen Gläsern aus der Dorfkirche in Koszewko (Polen)im Environmental Scanning Electron Microscope (ESEM) mit EDX. Die Ergebnisse zeigen, dass die Glasverwitterung schon sehr stark vorangeschritten ist und ein zukünftiger Schutz der mittelalterlichen Glasmalereifelder vor Umwelteinflüssen unbedingt notwendig ist. Es wurde ein Schutzverglasungsystem entwickelt, das speziell auf den langfristigen Erhalt der einzelnen mittelalterlichen Glasmalereifelder ausgelegt ist.
Specific co-fertilization of nutrients can enhance their plant-availability and thus the yield of plants. To investigate this effect, we performed a pot experiment with three different P-fertilizers and ammonium nitrate sulfate as a co-fertilizer, without and with a nitrification inhibitor (NI), and analyzed the form of nitrogen (N) in the soil via novel X-ray spectroscopic method. The application of NI with the N fertilizer led to a higher dry matter yield of maize. Novel N K-edge micro-X-ray absorption near-edge structure (micro-XANES) spectroscopy identified that the application of a NI promotes the temporary formation of a non-exchangeable N in detectable hot-spots in the soil. The subsequent slow release and prolonged availability of N during plant growth leads to higher yield. It can be concluded that NIs lead to a temporary fixation of ammonium-N in a pool that can be accessed by plant roots. Those types of available nutrient pools meet the idea of so-called “next generation fertilizers” as plants have access to nutrients according to their current demand.
Phosphorus (P) resource availability is declining and the efficiency of applied nutrients in agricultural soils is becoming increasingly important. This is especially true for P-fertilizers from recycled materials which often have a lower plant-availability compared to commercial P-fertilizers but are expected to play an increasingly important role into the future (Kratz et al. 2019). One promising way to increase the plant-availability of the fertilizer P is a co-fertilization with specific nitrogen (N) forms which can enhance the P uptake and make P-fertilizers from recycled material more competitive to commercial phosphate rock-based P-fertilizers (Rahmatullah et al. 2006; Vogel et al. 2018). To investigate this effect, we performed a pot experiment with three different P-fertilizers (sewage sludge-based, phosphate rock and triple superphosphate) and ammonium nitrate sulfate as a co-fertilizer, without and with a nitrification inhibitor (NI), and analyzed the form of N and P in soil via a suite of chemical and novel X-ray spectroscopic methods. The application of NI with the P and N fertilizers led to a higher dry matter yield and a higher P uptake of maize. Novel N K-edge micro-X-ray absorption near-edge structure (micro-XANES) spectroscopy identified that the application of a NI promotes the temporary formation of a non-exchangeable N in detectable hot-spots in the soil. The subsequent slow release and prolonged availability of N during plant growth leads to higher yield and nutrient uptake. It can be concluded that NIs lead to a temporary fixation of ammonium-N in a pool that can be accessed by plant roots. Those types of available nutrient pools meet the idea of so-called “next generation fertilizers” as plants have access to nutrients according to their current demand.
The halophilic bacterium Halomonas elongata can tolerate salt concentrations above 10% NaCl and uses the accumulation of the compatible solute ectoine as a major osmoregulatory mechanism. Ectoine can be accumalted inside the cell through import from the medium or de novo synthesis and establishes an osmotic equilibrium with the surrounding1. Ectoine also protects proteins from the effects of freezing, drying and high temperatures4 and DNA from ionizing radiation2. These features make ectoine a valuable compound for cosmetics and medical devices.
H. elongata was originally isolated from a solar salt facility, where it thrives under high salt concentrations. It was found that marine prokaryotes, which are exposed to high oxidative stress in their environment, vary glycolytic strategies5. A variation in the use of the glucose metabolic pathways is also assumed for H. elongata.
Introduction: Effective disinfection is crucial to maintain hygiene and to prevent the spread of infections. Phenotypic heterogeneity in disinfection survival (i.e. tolerance) may result in failure of disinfection, which in turn may foster the evolution of resistance to both disinfectants and antibiotics. However, the consequences of phenotypic heterogeneity for disinfection outcome and resistance evolution are not well understood. Goal: This study investigates the impact of phenotypic heterogeneity on the survival and evolution of Escherichia coli during disinfection with six commonly used substances. Furthermore, the consequences of evolved disinfectant tolerance for antibiotic resistance evolution are studied. Materials & Methods: The extent of population heterogeneity during disinfection is derived by determining time-kill kinetics and analysis with mathematical modelling. The link between population heterogeneity and evolvability of disinfectant tolerance was assessed by laboratory evolution experiments under periodic disinfection. The ability of disinfectant tolerant strains to evolve antibiotic resistance is assessed by serial transfer experiments with increasing concentrations of different antibiotics and by whole genome sequencing. Results: Multi-modal time-kill kinetics in three of the six disinfectants suggest the presence of disinfectant-tolerant subpopulations (i.e. persister cells). Importantly, the ability and extent to evolve population-wide tolerance under periodic disinfection is related with the presence of persister cells and the level of phenotypic heterogeneity during disinfection. Interestingly, the probability of high-level resistance evolution to certain antibiotics is attenuated in disinfectant tolerant strains as compared to the sensitive ancestor. Whole-genome sequencing reveals epistatic interactions between disinfectant tolerance and antibiotic resistance mutations, preventing access to canonical evolutionary paths to resistance. Summary: Our findings suggest that phenotypic heterogeneity can facilitate disinfection survival and the evolution of population wide tolerance, which can impact future antibiotic resistance evolution.
Up to now, different analytical methods for single cell analysis exist focusing on key features such as size, shape, morphology and elemental composition. The combination of the latest ICP-MS techniques - ICP-ToF-MS - together with the latest developments in the field of sample delivery - micro droplet generator (MDG) – will allow a Deep insight into the composition and size of cells.
Microbiologically influenced corrosion (MIC) is an oxidation of metals affected by the presence or activity (or both) of microorganisms e.g. Shewanella Putrefaciens in biofilms on the surface of the corroding material. As this can happen for example in the soil on iron pipes of water pipes, in oil tanks or on steel sheet piling, there is great interest in MIC research, not only from various industrial sectors, but also from the environmental aspect.
Infections with multi-resistant Gram negative pathogens are a major threat to our health system. In order to serve the needs in antibiotics development we selected untapped bioresources and implemented high throughput approaches suitable for the discovery of strains producing antibiotics with anti-Gram negative activity.
Our approaches rely on the hypothesis that Termite associated bacteria are likely to produce potent antibiotics to defend their hosts against entomopathogenic microorganisms. Termite nests and guts harbor suitable, highly diverse microbiomes in which bacterial taxa are present known to potentially produce natural compounds. In
a first step the diversity of Coptotermes species nest microbiomes was assessed carefully by using 16S rDNA amplicon sequencing on the Illumina MiSeq platform and nest material was selected to retrieve viable cells by using Nycodenz density gradient centrifugation. In order to analyze the diversity of the culturable termite nest microbiome, bacterial cells were either distributed in 384-well plates
(approach 1) or encapsulated in small spheric agarose beads by an high throughput microfluidics technique (approach 2). Cultures obtained from approach 1 were scaled-up in 96-well Duetz-systems for characterization of diversity and for rapid supernatant screening using the bioluminescence-labeled E. coli pFU166. The generated
droplets of approach 2 simultaneously received a small population of GFP-tagged Gram negative screening cells and were sorted for low fluorescence using FACS. After elimination of redundancy we performed a fast scale-up of active strains.
Implementation of this pipeline allows us to prioritize antibiotics producing strains in a ultra-high throughput fashion and by cultivation of broad diversity in our approches.
Near-ambient pressure XPS makes it possible to characterise samples not compatible to ultra-high vacuum, and enables the study of liquid-solid, gas-liquid and gas-solid interfaces. NAP-XPS meas-urements of biofilms, suspended nanoparticles and metal-organic frameworks were performed with EnviroESCA developed by SPECS.
An interesting application is surface characterisation of biofilms, which are bacterial communities embedded in a self-produced polysaccharide matrix. Various model systems ranging from pure polysaccharides of alginate to biofilms harvested directly from the growth medium have been char-acterised in humid conditions[1].
NAP-XPS also makes it possible to characterise nanoparticles in solution. Silver nanoparticles in aqueous solution were characterised and the Ag 3d-spectrum compared to spectra obtained of dried nanoparticles in UHV-conditions[2]. The binding energy of the Ag 3d-core level peak was shifted by 0,6 eV towards higher binding energy for suspended nanoparticles compared to the dried sample measured in UHV. This can be assigned to a change in surface potential at the water-nanoparticle interface.
Metal-organic frameworks (MOFs) are suitable materials for gas storage of small molecules due to their nanoporous, crystalline structure. However, instability in humidity remains an issue for many types of MOFs. XPS-measurements of the MOF-structure HKUST-1 were performed in various NAP-conditions to assess the stability of the sample and its interaction with the gas molecules as water, methanol and pyridine.
Black yeasts form a polyphyletic group of Ascomycota that colonize bare surfaces like rocks, facades of buildings, and solar panels. Their protective adaptations enable an adequate response to fluctuating and diverse temperature, water and UV radiation stresses. Together with bacteria and algae they form sub-aerial biofilms (SAB) this way discoloring and weathering the surfaces they grow on. Strain A95 of Knufia petricola (Eurotiomycetes, Chaetothyriales) displays both typical yeast-like cell growth and constitutive dihydroxynaphthalene (DHN) melanogenesis. Along with the cyanobacterium Nostoc punctiforme as photobiont, it is already used in a model system for studying SAB formation and bio-weathering. Applying the recently developed tools for the generation of deletion mutants will allow to define gene functions and to identify genes critical for abiotic and biotic interactions. We present a chromosome-level genome assembly and annotation for K. petricola A95. The genome was assembled with MaSuRCA using a hybrid assembly approach of Illumina MiSeq and PacBio SMRT sequencing data. The resulting assembly consists of 17 contigs including the complete mitochondrial genome and five complete chromosomes. It shows indication of repeat-induced point mutations (RIP). Supported by RNA sequencing data from eight different growth conditions, 10,994 genes were predicted with the BRAKER2 pipeline. Functional annotation of genes was obtained from general functional annotation databases and the fungal specific database FungiPath. Comparative analyses are in progress to identify genes specific to black yeasts, that may facilitate the survival on exposed surfaces. In sum, the genome sequence of K. petricola is a valuable resource to gain insight into the protein inventory and functional pathways of extremotolerant and symbiosis-capable fungi.
First insights into Chinese reverse glass paintings gained by non invasive spectroscopic analysis
(2019)
A non-invasive methodological approach (X-ray fluorescence (XRF), diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), Raman spectroscopy) has been carried out to identify the pigments and classify the binding media in two Chinese reverse glass paintings (The Archer, Yingying and Hongniang) from the late 19th and early 20th centuries. The results reveal a combined use of traditional Chinese and imported European materials. Several pigments like cinnabar, lead white, orpiment, carbon black and copper-arsenic green (probably emerald green) were found in both paintings; red lead, artificial ultramarine blue, Prussian blue and ochre appear in at least one of the paintings.
The presence of portlandite (Ca(OH)2) along calcite (CaCO3) in the fine-grained, white backing layer of Yingying and Hongniang indicates the presence of limewash. In Chinese tradition, limewash was produced from clamshells, and was then sold as clamshell white. In contrast to the Japanese pigment, Chinese clamshell white was made of finely grounded shells, which were heated over a low fire. The residue (CaO) forms portlandite (Ca(OH)2) when water is continuously added. This water-rich mixture is applied on the painting. Portlandite reacts with atmospheric CO2 during drying and forms fine-grained calcite (CaCO3) [1,2].
The identification of emerald green (The Archer) suggests an earliest manufacturing date in the 1830s [3] and promotes the sinological dating of the painting. Drying oil was classified as a binding media in most areas of both paintings. However, the orange background of The Archer yielded prominent bands of both proteinaceous and fatty binder.
Black fungi belong to different classes of Ascomycota but evolved similar morpho-physiological adaptations such as yeast-like growth and constitutive melanin formation to colonize extreme competition-free environments. They are ubiquitously found on air-exposed surfaces, from ancient marble monuments to modern solar panels. The rock inhabitant Knufia petricola was chosen to become a model for these extremotolerant black fungi. Plasmid-based and ribonucleoprotein-based CRISPR/Cas9 techniques were introduced to precisely introduce one to multiple double strand breaks into the DNA to modify, replace or add sequences to the genome either using the available selection marker systems (hygR, natR, genR, baR, suR) or by marker-free approaches. Multiplexing is very efficient, allowing for four or more simultaneous genome editing events. The newly generated cloning vectors containing the Tet on construct for doxycycline-controlled gene expression, and the validated sites in the K. petricola genome for color-selectable (pks1, phs1, ade2) or neutral insertion (igr1 to 5) of expression constructs complete the reverse genetics toolbox. One or multiple endogenous or heterologous genes can be expressed on demand from different genomic loci or from a single construct by using 2A self-cleaving peptides. We thus can express genes from synthetic clusters in a non-pigmented background (Δpks1/Δphs1). The fact that K. petricola only produces few secondary metabolites (DHN melanin, carotenoids, mycosporines and a siderophore) and plant cell-wall degrading enzymes but is capable to take the burden of acetyl-CoA-consuming metabolism and protein secretion renders K. petricola a promising host for the expression of heterologous genes encoding high-end secondary metabolites and enzymes.
Fungal biofilms on materials: describing and modelling growth of the black fungus Knufia petricola
(2023)
Fungi that grow as biofilms are associated with clinical settings as well as various cases of material fouling and material damage. Black fungi as biofilm formers have been rarely studied so far. Their conspicuous dark pigmentation, EPS production, adhesion capabilities and adaptations to stresses allow black fungi to develop biofilms on materials under harsh conditions. For example, rock-inhabiting black fungi withstand sun irradiation and dehydration and are therefore ubiquitous on arid surfaces like solar panels and marble monuments.
To understand and control their ability to colonise and deteriorate materials, one should assess and model black fungi’s growth patterns. But so far, no mathematical model has been developed to describe their growth. Knufia petricola A95, representing rock-inhabiting fungi from Chaetothyriales, is genetically amenable and can serve as a model for biofilm studies in black fungi. The primary objective of this project is to develop a growth model for K. petricola A95 which will enable to define and predict material colonisation of black fungi.
Dedicated experimental work with K. petricola will allow the quantitative assessment of the impact of environmental conditions (e.g. pH, nutrients, etc.) on the growth behaviour at the biofilm and single cells level. Data which will be used to validate and develop an individual-based model (based on the iDynoMICS modelling platform) that explains how fungal biofilms form, colonise materials, and cause deterioration.
Thus far, research has been conducted on the impact of different concentrations and sources of major elements (e.g. C, N, …), as well as trace elements (e.g. Cu, Mg, …), on the colony shape and biomass of Knufia petricola A95 biofilms. To study the behaviour of single cells, the length of the cell cycle in different growth media has been determined via the combined use of microfluidic devices and confocal microscopy.
Gypsum (calcium sulfate dihydrate) has excellent building material properties and has been widely used in constructions in the last decades in many countries. Accordingly, an increase of waste gypsum in C&D waste is expected in the upcoming years. On one hand, sulfates are unwanted in other secondary building materials (particularly in recycled concrete aggregates) and should be minimized for quality reasons. On the other hand, used gypsum from CDW can also be used in gypsum production if the high quality requirements for the recycled gypsum – especially regarding the sorting accuracy - are met.
A large percentage of the gypsum from buildings was installed as gypsum plasterboards in interior fittings so far. Gypsum plasterboards are comparatively simple to remove and to separate during selective dismantling. Therefore, a high sorting purity can be achieved. In addition, techniques for the recycling of gypsum plasterboards already exist and high quality standards can be achieved. Also, the reuse in gypsum production has been improved in the last decade. Furthermore, an environmental evaluation of the whole process of gypsum plasterboard recycling and reuse showed that this approach can be environmentally advantageous. Therefore, a closed-loop recycling of gypsum plasterboards is feasible.
This poster will show the development of gypsum consumption in different countries as well as a prognosis for the upcoming of gypsum in CDW in the future decades in Germany. Furthermore, a simplified scheme of the recycling process and selected results from an environmental evaluation will be presented.
The global rise of antibiotic resistance has made the proper use of disinfectants more important than ever. Their application in clinical l settings is an integral part of antibiotics stewardship by preventing the occurrence and spread of infections. However, improper use of disinfectants also harbours the risk for the evolution of tolerance and resistance to disinfectants, but also to antibiotics. It is therefore crucial to understand whether and how bacteria can survive chemical disinfection and which conditions facilitate the evolution of tolerance and resistance.
Here, we study the heterogeneity in the response of isogenic E. coli populations exposed to different levels of commonly used disinfectants. At concentrations below the minimal inhibitory concentration (MIC), we find that certain disinfectants induce prolonged lag times in individual cells, a phenotype that has been associated with persistence against antibiotics. At concentrations above the MIC, we find heterogeneous killing for a range of the tested substances. Interestingly, for the three cationic surfactants that were tested, we find kill kinetics revealing the presence of a tolerant subpopulation that can withstand disinfection longer than most of the population. We will present results from an ongoing evolution experiment in which we test the potential for evolution of population-wide tolerance and resistance through intermittent exposure to lethal doses of a cationic surfactant.
The exposure to antimicrobial substances drives the evolution of antimicrobial resistance (AMR). Biocides are antimicrobials used as disinfectants, antiseptics and preservatives. They find application on a large scale in the industrial and medical sector, but also in private households. In terms of mass, the worldwide use of biocides exceeds that of antibiotics. Thus, despite their important role in preventing the spread of pathogens, due to their ubiquity, biocides are suspected to be drivers of the antimicrobial resistance crisis. In our work we try to understand how biocides contribute to the emergence of AMR, what the underlying adaptation principles and mechanisms are and how they compare to those found for antibiotics.
Here, I present results which demonstrate that phenotypic heterogeneity in E. coli, namely the formation of tolerant persister cells, can enable the survival of disinfection and consequentially facilitate the evolution of AMR. Using experimental evolution that selects for tolerance, we find that mutations in lipid A biosynthesis arise following periodic treatment with benzalkonium chloride (BAC), a widely used disinfectant. BAC tolerance is associated with additional, diverse mutations as well as changes in the susceptibility to antibiotics from different classes and changes in motility and biofilm formation, suggesting extensive regulatory re-wiring in the evolved clones. Remarkably, we find that fitness costs (growth rate reduction) incurred by BAC tolerance are alleviated in the presence of antibiotics. These findings demonstrate the complexity underlying the adaptation to antimicrobials and highlight the links between persistence to disinfectants and resistance evolution to antimicrobials.
Besides classical per- and polyfluorinated alkylated substances (PFAS), side-chain fluorinated polymers (SFPs) are widely applied as efficient anti-wetting and anti-greasing coatings in various daily applicated consumer products such as outdoor apparel, carpetry or paper-based food contact materials. Although the fluorinated sidechains are chemically bound to a base polymer, their release can be triggered by environmental influences or wear-off, thus contribute as significant sources of PFAS in the environment. Since only little knowledge of PFAS and SFP composition in consumer products is available, a comprehensive analytical approach might be beneficial.
Surfaces of metallic copper and copper alloys effectively inactivate microorganisms and viruses. However, the exact inactivation mode is still under debate. Main factors are assumed to include direct contact with the metallic surface, influx of Cu(I)/Cu(II) ions and the generation of reactive oxygen species (ROS). Laser-induced periodic surface structures (LIPSS) are frequently reported to act antibacterial, mainly by prevention of bacterial adhesion due to a limited number of possible adhesion points or by increasing the overall surface of intrinsically antibacterial materials. In time-kill experiments with E. coli and S. aureus we analyzed the impact of LIPSS on the toxicity of metallic copper and brass. We also conducted ROS accumulation assays and conclude that the application of LIPSS is not generally straight forward to obtain or improve antibacterial surfaces. Thus, the antibacterial effects of LIPPS.
The technique of painting on the reverse side of glass was rediscovered by artists in the early 20th century. The artist group “Der Blaue Reiter” around Wassily Kandinsky and Franz Marc got in touch with this technique in 1908 and 1909. In the following years it gained great popularity, especially in Germany. Nevertheless, the technique has not received its due appreciation in art history. It was considered as stained glass. However, the paint layers are applied cold, hence this artistic technique doesn’t involve a firing step.
Our multidisciplinary project investigates the art historic backgrounds, the painting techniques and materials of modern reverse paintings on glass. More than 1000 paintings from ~100 artists were discovered in the framework of our project. A selection of 60 paintings could be analyzed using non-invasive, in-situ methods such as Raman and VIS spectroscopy, Diffuse Reflectance Fourier Transform Infrared Spectroscopy (DRIFTS) and X-ray fluorescence (XRF). In this paper we want to point out the key role of Raman spectroscopy for our research. It offers the unique opportunity to measure paint layers from both sides. (front = through the glass; reverse = directly on the paint layer).
Inter-pulse accumulation of heat could affect the chemical and morphological properties of the laser processed material surface. Hence, the laser pulse repetition rate may restrict the processing parameters for specific laser-induced surface structures. In this study, the evolution of various types of laser-induced micro- and nanostructures at various laser fluence levels, effective number of pulses and at different pulse repetition rates (1 – 400 kHz) are studied for common metals/alloys (e.g. steel or titanium alloy) irradiated by near-infrared ultrashort laser pulses (925 fs, 1030 nm) in air environment. The processed surfaces were characterized by optical and scanning electron microscopy (OM, SEM), energy dispersive X-ray spectroscopy (EDX) as well as time of flight secondary ion mass spectrometry (TOF-SIMS). The results show that not only the surface morphology could change at different laser pulse repetition rates and comparable laser fluence levels and effective number of pulses, but also the surface chemistry is altered. Consequences for medical applications are outlined.
Microbiologically influenced corrosion is a multidisciplinary research area. To develop successful mitigation strategies, expertise from the industry and research institutes are essential. In Department 4.1, we developed an innovative laboratory flow model (Hi-Tension) that allows effective monitoring of MIC under both standard and non-standard conditions. The flow model allows flexibility with material selection, flow rates, temperature and other environmental parameters changes. Furthermore, the flow model allows integration of electrochemical measurements using microsensors, providing a comprehensive view of corrosion at the biofilm level. Currently, initial results indicate corrosion in the flow model is significantly higher than that of standard laboratory set ups, i.e. static incubations, particularly for methane-producing microorganisms.
Microbiologically influenced corrosion (MIC) is the deterioration of metals due to the metabolic activities of microorganisms. Microorganisms can take electrons directly from the metal surface (EMIC) thereby causing corrosion. Well known culprits of EMIC are: sulfate-reducing bacteria (SRB), acetogens and methanogens.
Microbiologically influenced corrosion (MIC) is the deterioration of metals due to the metabolic activities of microorganisms. Microorganisms can take electrons directly from the metal surface (EMIC) thereby causing corrosion. Well known culprits of EMIC are: sulfate-reducing bacteria (SRB), acetogens and methanogens.
Our aims
- Develop a novel flow system to study MIC by methanogens to mimic industrial Environments
- Investigate the inhibitory concentrations of biocides targeting SRB on corrosive methanogenic strains
- Investigate the inhibitory effects of corrosion inhibitors on methanogens
- Compare the inhibitory concentrations to SRB
Microbially influenced corrosion (MIC) of iron is usually attributed to sulfate-reducing microorganisms (SRM) which act upon the metal by the reactiveness of hydrogen sulfide, and by withdrawal of the available electrons in electrical contact with the metal. Methanogenic archaea can also cause MIC by directly withdrawing electrons from the iron surface for methanogenesis. However, the mechanistic details and kinetics of the overall process are poorly understood. Precipitation of siderite, a by-product of methanogenesis, (4Fe + 5HCO3 + 5H+ 4FeCO3 + CH4 + 3H2O) can lead to an insulating layer on the metal surface and lower the corrosion rate. Still, the extent of FeCO3 precipitation may be significantly influenced by environmental conditions such as pH and advective processes.
Per- and polyfluoralkyl surfactants (PFAS) are industrially produced surface chemicals used in daily applications that have gained public and political attention due their unnaturally high appearance in drinking water, nourishments and soils.[1] Their chemical structures exhibit both lipophilic and hydrophilic properties, leading to a highly inert and persistent character.[2] Various PFAS have shown to be bioaccumulative in plants and animals, and some have been characterized as highly toxic when ingested.[3] When exposed to the environment, PFAS slowly get mobilized by natural water resources, leading to contamination of large areas of soil and natural water sources.[4] While PFAS contamination of drinking water has been investigated intensively, perfluoralkyl contamination of soils has been rarely examined as of yet. At the same time, an increasing amount of PFAS contamination sites are being discovered worldwide, calling for a sophisticated strategy towards analytical characterization.[5] Since the number of known PFAS already exceeds 4700, the established sum parameters like extractable organic fluorine (EOF) and adsorbable organic fluorine (AOF) are key elements to fully survey the impact of exposure.[6] Simultaneously, innovative soil remediation strategies are required to contain environmental destruction and to minimize further spreading of contaminants.[7] Addressing these challenges requires suitable analytical devices that are capable of mobilizing PFAS in the solid as well as in the liquid phase. The use of combustion ion chromatography (CIC) enables analysis of both immobile and volatile PFAS and allows the detection of both total fluorine (TF) and EOF or AOF of a given soil or aqueous sample. Based on preliminary results, the clean-up of a PFAS contaminated matrix is monitored via EOF detection over time. Additionally, we tested the effectiveness of urea (CH₄N₂O) as fluoride scavenger with the aim to improve the separation of inorganic and organic fluorine and therefore, to improve AOF accuracy. The presented findings can be contributive with regard to future AOF/EOF sum parameter development and application.
Since the early 19th century microorganisms were studied on their capabilities of causing microbiologically influenced corrosion (MIC) of metals. The most studied ones are sulfate-reducing bacteria (SRB), but others can corrode metals as well, e.g. acid-producing bacteria or methanogenic archaea (MA). However, these studies were mostly focused on metals related to the petroleum industry but metals for other industries, e.g. dentistry, are also susceptible to corrosion. The inert Titanium (Ti) is often used as an implant material, but it is a base metal. The formation of a passivating oxide layer allows Ti to be corrosion resistant at normal conditions.
Nonetheless, scanning electron microscope images on dental implants from patients with acute peri-implantitis showed clear signs of corrosion. Currently, the corrosion mechanism of dental implants is unknown, but many indications suggest that oral microorganisms, including MA (Methanobrevibacter oralis) and SRB (Desulfomicrobium orale), could be involved.
To determine if MA or SRB can corrode Ti (pure Ti or Ti-6Al-4V alloy), corrosion rate, methane and sulfide concentrations were analyzed. Electrical potential measurements using in-house developed electrochemical cells indicated a potential change on Ti in the presence of a corrosive MA strain compared to an abiotic control.
Microbial composition comparison will be analyzed using samples from dental pockets of 150 infected patients by considering the quality of the implant and 50 healthy people by means of amplicon sequencing. Enrichments and isolation of pure cultures from the dentals samples are also examined for their corrosion behavior. Overall, this is the first study investigating the susceptibility of dental implant material to corrosion using human related MA.
There is a need for an assessment of the emission properties of volatile organic compounds (VOCs) from consumer products. A method comparison was carried out to evaluate adapted and cost-effective procedures for such items. Smaller and automated emission chambers de-picted similar kinetics compared to a 203 L standard chamber. Toy samples made of PVC (Polyvinyl chloride) emitted more VOCs compared to other tested polymeric products. The emissions from 2 selected samples were studied to allow an evaluation of the resulting room concentration and external exposure of a child. Obtained concentrations were not of concern.
Iron is arguably the most essential metal in living organisms. For rock-inhabiting fungi its acquisition might be unconventional as they (1) tend to inhabit iron-deficient, oxygen-rich surfaces like marble monuments and solar panels and (2) produce the black, iron-adsorbing pigment melanin. We used a range of analytical methods, ongoing mineral dissolution experiments and gene deletion mutants of the model rock-inhabiting fungus Knufia petricola to figure out the mechanisms and substrate deteriorating effects of iron uptake by these organisms. To study both siderophore-mediated and reductive iron assimilation (RIA), genes like sidC, encoding a putative siderophore synthetase and ftr1 and fet3 encoding the subunits of an iron permease-oxidase were deleted.
At iron deficient conditions, growth of the wild type (WT) and ΔsidC mutant was similar, whereas growth of the Δftr1-fet3 mutant and the double mutant ΔsidC/Δftr1-fet3 was diminished and absent, respectively. We were not able to detect the siderophore of K. petricola and the WT and mutants were not able to grow at low concentrations of strong iron chelators. However, in a cross-feeding experiment, an overexpression strain of sidC allowed more growth of ΔsidC/Δftr1-fet3 on iron deficient medium than the WT, whereas the ΔsidC mutant could not do so at all. Compared to the WT, the sidC overexpression strain also withstood oxidative stress better and had a shorter lag time and higher growth rate. Combined, these results indicate that K. petricola relies more on RIA than siderophore-mediated uptake as it likely excretes low quantities of a primarily intracellular siderophore. Interestingly, Δftr1-fet3 had a higher iron content than the WT at iron deficient conditions. This difference disappeared upon deletion of melanin synthesis (Δpks1 vs. Δpks1/Δftr1-fet3): melanin-bound iron can likely not be used without RIA. K. petricola’s chelation incapacity implies a habitat free of competition for iron while offering us a mitigation strategy.
We demonstrate a 2D platform based on high contrast wetting patterns suitable for miniaturized microbiological assays.
In principal, superhydrophilic spots are surrounded by a superhydrophobic surface area. The special structure of the superhydrophilic functional surface ensures that liquids, e.g. bacterial suspensions or biocide solutions, spread immediately and evenly on this surface without passing the wetting boundary. This feature allows a homogenous distribution of bacteria or chemical substances on well defined lateral dimensions. The superhydrophilic spots may also serve as substrate for bacterial biofilms. Due to the high wetting contrast and the fabrication process, it is possible to minimize the test areas as well as their distance to each other.
We demonstrate the fabrication process of the high wetting contrast platform and also present a microbiological assay as an application example. Advantages of this platform are the use of low volumes and its potential of automated analysis.
Leachiing tests - a useful tool for the environmental impact assessment of construction products?
(2018)
Construction products and waste materials used for construction can be in contact with the environment and may release potential harmful compounds. Information on the total content of these substances in the product is not sufficient to assess its envrionmental impact since it does not consider realistic exposure conditions.The impact of these compounds is often assessed by using the total content in the product. This estimation lacks realistic exposure conditions.
Concerning the pathway to soil and groundwater by contact with rain or seepage water leaching tests, which were developed and standardized by the European Committee for Standardization, are available. While for secondary construction products and waste this investigation of the leaching behaviour is standard procedure and is already part of regulation in Germany (draft ordinance on reuse of mineral waste) and within Europe (landfill directive) the release from primary construction products got in the focus more recently. Several monitoring studies found unexpected high concentrations of substances used as herbicides and fungicides in surface water and stormwater originating from urban areas. As some of these compounds are even banned for the use in agricultural applications in Europe alternative sources as roof materials and façade coatings exposed to rain were suggested. Further field and laboratory tests confirmed construction products as sources of these substances in water.
The aim of this presentation is to show exemplary results of existing leaching methods and underline the strength and weaknesses of the test system with selected examples from our work. We especially draw the attention to the research which is still needed to close the gap between the results of leaching experiments and the subsequent risk assessment of the products.
Bottom ash (BA) from municipal solid waste incineration (MSWI) contains harmful substances such as heavy metals, chloride and sulfate which are mobilized in contact with water. Standardized leaching tests are used to measure the extent of mobilization. It is known that fresh bottom ash displays elevated concentrations of various heavy metals such as lead or zinc due to the formation of hydroxo complexes as a result of high pH values of 12 and above. Storage of BA is accompanied by ageing processes, mainly the reaction of CaO and Ca(OH)2 with CO2 leading to lower pH values in contact with water around 11. Usually heavy metals concentrations are minimum at these conditions. Knowledge of the long-term leaching behavior of potentially harmful substances is crucial for the assessment of the environmental compatibility of reusing municipal solid-waste incineration bottom ash (MSWI BA) in construction, i.e., as a road base layer. BA fractions obtained from wet-processing aiming at the improvement of environmental quality were used to investigate the mobility of relevant substances. Eluates from laboratory-scaled leaching procedures (column percolation and lysimeters) were analyzed to learn about the long-term release of substances. Unsaturated conditions and artificial rainwater were used in the lysimeter tests to simulate field conditions. In addition, batch test eluates were generated at usual liquid-to-solid ratios (L/S) for compliance testing purposes. A variety of cations and anions was measured in the eluates. The wet treatment reduces the leaching of chloride and particularly sulfate by more than 60%. The release of typical contaminants for the treated MSWI BA such as the heavy metals Cu and Cr was well below 1% in the conducted leaching tests. An increase in the Sb concentration was observed in the lysimeter experiments starting at L/S 0.75 L/kg and in the column experiment at L/S 4 L/kg is assumed to be related to decreasing concentrations of Ca and thus to the dissolution of sparingly soluble calcium antimonate. The same leaching mechanism applies with V, but the concentration levels observed are less critical regarding relevant limit values. However, on the long term the behavior of Sb could be problematic for the application of MSWI BA as secondary building material.
Brominated flame retardants have widely been used for more than 50 years and have been frequently detected in environmental samples as surface water and fish. Leaching from construction products was identified as one possible source. The use of substances as e.g. hexabromocyclododecane (HBCCD) was restricted recently due to increasing concern about negative impacts on the environment and human health caused by the persistent, bio accumulative and toxic properties. New polymeric brominated flame retardants have been developed to replace HBCCD in expanded polystyrene (XPS), which is used for insulation of buildings. It is assumed that the release from the polymer is much smaller in comparison to release of small molecules as HBCCD. The release from the polymer may include monomers or small fragments of different size. Thus, a sum parameter is needed. According to Schlummer et al. it is possible to detect brominated molecules using X-ray fluorescence spectroscopy by determination of brome content in XPS extracts. As only small brominated compounds can be extracted it also allows for the differentiation between the two classes of compounds. This method was applied to verify the presence of different kinds of flame retardants in XPS samples and to evaluate the leaching behavior.
Microbiologically influenced corrosion (MIC) of iron is usually attributed to sulfate-reducing microorganisms, either chemically (formation of hydrogen sulfide) or electrically (direct electron uptake). Methanogenetic Archaea are also known to be involved in iron corrosion, forming a multi-species biofilm on corroding metallic structures. However, mechanistic details and kinetics of the overall process in methanogen-induced MIC are poorly understood.
Microbially influenced corrosion (MIC) of iron is usually attributed to sulfate-reducing microorganisms (SRM) which act upon the metal by the reactiveness of hydrogen sulfide, and by withdrawal of the available electrons (Fe → Fe²⁺ + 2e⁻ ; E° = 0.47 V) in electrical contact through surface attachment. Also methanogenic archaea are supposed to cause MIC. Because they do not produce hydrogen sulfide, withdrawal of electrons may be their main corrosive mechanism; however, mechanistic details and kinetics of the overall process are poorly understood. Precipitation of siderite (4Fe + 5HCO₃⁻ + 5H⁺ → 4FeCO₃ + CH₄ + 3H₂O) can lead to an insulating layer on the metal surface and lower the corrosion rate. Still, the extent of FeCO₃ precipitation may be significantly influenced by environmental conditions such as pH and advective processes.
To investigate the corrosive potential of methanogens, we studied strains isolated from marine sediments (Methanococcus maripaludis 14266, 2067, Methanobacterium-affiliated strain IM1), crude oil tanks (Methanococcus maripaludis Mic1c10, KA1) and the oral cavity (Methanobrevibacter oralis) in a closed (batch) culture, and in a sand-packed flow-through cell with pH control and simulation of a fluctuating environment. Results indicate that the rates of iron corrosion due to coupled methanogenesis (up to 0.3 mm/yr) are comparable to that caused by SRM. Surface analyses of the metal showed severe pitting. Such knowledge and deeper understanding also from an electrokinetic point of view may not only provide further models in microbial electrophysiology, but also contribute to mitigation strategies in MIC.
Microbially influenced corrosion (MIC) of iron is usually attributed to sulfate-reducing microorganisms (SRM) which act upon the metal by the reactiveness of hydrogen sulfide, and by withdrawal of the available electrons in electrical contact with the metal. Methanogenic archaea are supposed to cause MIC. Because they do not produce hydrogen sulfide, withdrawal of electrons may be their main corrosive mechanism; however, mechanistic details and kinetics of the overall process are poorly understood. Precipitation of siderite (4Fe + 5HCO3 + 5H+ 4FeCO3 + CH4 + 3H2O) can lead to an insulating layer on the metal surface and lower the corrosion rate. Still, the extent of FeCO3 precipitation may be significantly influenced by environmental conditions such as pH and advective processes.
Microbially influenced corrosion (MIC) of iron is usually attributed to sulfate-reducing microorganisms (SRM) which act upon the metal by the reactiveness of hydrogen sulfide, and by withdrawal of the available electrons in electrical contact with the metal. Methanogenic archaea are supposed to cause MIC. Because they do not produce hydrogen sulfide, withdrawal of electrons may be their main corrosive mechanism; however, mechanistic details and kinetics of the overall process are poorly understood. Precipitation of siderite (4Fe + 5HCO3 + 5H+ 4FeCO3 + CH4 + 3H2O) can lead to an insulating layer on the metal surface and lower the corrosion rate. Still, the extent of FeCO3 precipitation may be significantly influenced by environmental conditions such as pH and advective processes.
Microbial modelling of sulfate-reducing bacteria (SRB) and methanogenic archaea (ME) using iron
(2019)
Sulfate reducing bacteria (SRB) and methanogenic archaea (MA) are commonly found in the oil and gas environments. The formation of hydrogen sulfide (HS-) is particularly concerning for the petroleum industry due to its corrosiveness. However, the activities of SRB are limited to the concentration of sulfate present in the environment, whereas methanogens can utilize substrates such as H¬2 for methanogenesis. MA is commonly found in sulfate-free environments, such as deep sediments, and are known to form interspecies electron transfer relationships with SRB. Recently, SRB and MA capable of microbiologically influenced corrosion (MIC) by using elemental iron as a direct electron source (EMIC) have gained increased attention. On the iron surface, EMIC-SRB can outcompete EMIC-MA in the presence of sulfate, but this changes as sulfate depletes. The formation of FeS on the metal surface can be further utilized by MA for methanogenesis as it provides a conductive path. However, the possible kinetics involved of the overall process are currently unknown.
We obtained a co-culture of EMIC-SRB and EMIC-MA to investigate the growth rates and electrical potential changes under different environmental conditions, including changes in pH, temperature and salinity. Results indicate that under neutral conditions and using iron as the sole substrate, methane production (up to 5 mM) starts after sulfate was depleted. Electrochemical measurements will be conducted on the co-culture under different conditions to determine the changes in the electrical potential in correlation with the sulfate and methane concentration. Fluorescence and electron microscope images of the biofilm structure will be used to visualize cell distribution and morphology. This study embarks the first step of understanding the relationship between EMIC-SRB and EMIC-MA. Such knowledge is important for the field of microbial electrophysiology and can be further explored for industrial applications.
Initial situation:
Aluminum is an often-used building material in modern architecture, not only for construction but as well for facades and decorative elements. In the 1950th and 1960th, after World War II, many buildings in Germany were constructed with aluminum or contain elements of colored anodized aluminum. In the last years a larger number of these buildings are increasingly in the sight of conservation works including the aluminum parts such as window frames or facade coverings.
Damaged Aluminum Surfaces:
Common damages are a change of color or gloss changes through weathering processes, drill holes or marks due to later modifications, scratches in the anodized layer due to extensive wear e.g. at handrails or door handles.
To repair damaged aluminum surfaces, there are usually two options: smaller damaged areas are repaired by using a touch-up pen. In case of larger damages, the complete re-anodization is necessary. This includes to de-anodize the surface with cleaning and grinding the whole aluminum object. Both possibilities are disadvantageous for the objects. The touch-up pen often does not match the color of the original surface together with an insufficient corrosion protection for outdoors. While the newly anodized surface differs in color and gloss from the originally applied color.
Research Approach:
The whole procedure contrasts with the principal approach in conservation which aims to intervene as less as possible, in case of the conservation of an object. To fulfill this approach in a more appropriate way the research project focuses on a mobile and partial application for colored, anodized aluminum parts.
To anodize aluminum the application of an electrolyte onto the surface together with sufficient voltage and current is necessary. Generally diluted sulfuric acid is used as electrolyte. Different possibilities are examined to enable the mobile application of the electrolyte, e. g. the application by producing a gel matrix or like in electroplating by pen or brush wrapped with a fleece fabric.
Experimental part:
First experiments are conducted to examine the structure of the anodized layer in relation with proper cleaning, anodization time with applied voltage and current and the coloring process. The aim was to reduce the preparation procedure and the anodization time as much as possible to facilitate the mobile application.
Examinations with Keyence microscope, Eddy current testing and REM are performed to characterize the layers. The results are shown in table 1. A clear connection between proper cleaning, anodization time, voltage and amperage and the achieved thickness of the anodized layer is significant. Cracks in the layer show that raising the voltage and amperage results in thicker layers but as well in a crumbled and less stable anodized surface.
Gel preparation:
In addition to the anodization process with a liquid e.g. sulfuric acid a gel application is tested to prevent the electrolyte from rinsing down during the mobile application. For this purpose, several gel-forming agents are tested together with their stability in acid systems. It was observed, that the consistency of the gels varies dependent of the time.
Conductivity:
The conductivity of sulfuric acid combined with different gel-systems was measured and compared in order to predict the possible growth of layers during anodic oxidation process.
Further steps:
Determination and optimization of application parameters like voltage, amperage and anodization-time to build up a preferably stable and sufficient thick anodized layer. Examination of gel preparation to guarantee a stable product, enforcing with textile tape for easy application.
One criterion to evaluate the odour of a sample is perceived
intensity П which is evaluated with the aid of a comparative scale. The procedure is well accepted but also time consuming and extensive. The study should help simplify the testing procedure by asking the panel members to only indicate whether a sample is perceived as less to equally intense or of greater intensity in comparison to a given acetone concentration of 7 pi on the comparative scale. In Germany a value of 7 pi is required for products to be suitable for the indoor use. The results show that the simplified test is basically suitable.
The new OECD test guideline will address the following four main steps in the determination of the length and width distributions of fibers: sample preparation, image acquisition, data evaluation and uncertainty analysis. As the sample preparation has to be optimized for each material, general quality criteria will be given in the protocol. For full visibility of a fiber the appropriate resolution has to be chosen. In the data evaluation the length and diameter of each fiber will be determined concurrently to allow for application of different regulatory definitions. The quality of the results critically depends on the sample preparation as well as the data evaluation. In this step the classification rules have to be formulated and followed accurately in order to optimize reproducibility of the method. The SOP will be validated in an international round robin test, which is planned for 2018/2019.
The OECD test guidelines (TGs) for testing chemicals have been widely used for regulatory purposes all over the world since the establishment of the Mutual Acceptance of Data (MAD) principle in 1984. This MAD principle ensures that, if a chemical is tested under the Good Laboratory Practice (GLP) conditions accordingly to an OECD TG, the data should be accepted in all OECD countries. The TGs have been developed, harmonized, internationally validated (round robin tests) and adopted by OECD countries to be used for the physical-chemical characterisation, fate estimation, and hazard identification for risk assessment of various chemicals. In addition to the TGs, OECD Guidance Documents (GDs) usually provide guidance on how to use TGs and how to interpret the results. These GDs do not have to be fully experimentally validated, and hence they are not under MAD, but they are based on relevant published scientific research.
But are the existing TGs and the related GDs applicable and adequate for the regulatory testing of nanomaterials? In general, it is accepted that most of the "endpoints" or more precisely measurement variables are applicable also for nanomaterials. However, for some endpoints new or amended TGs are needed. In addition, several GDs are needed to give more precise advice on the test performance in order to gain regulatory relevant data on nanomaterials.
The poster will present the status quo on recent TGs and GDs development for nanomaterials at OECD level with relevance for physical-chemical characterisation. Emphasis will be given to the proposed OECD TG on particle size and size distribution for manufactured nanomaterials. The development of such a TG is of special importance as particle size and size distribution is considered as major information for nanomaterial identification and characterization. A reliable and reproducible characterisation of particle size and size distribution is also needed for chemicals risk assessment of nanomaterials, for instance to interpret and compare test results and - in future – to forecast interaction and effects of nanomaterials. The presented poster will illustrate the way from the idea for a new TG and new GD to an accepted OECD TG/GD.
Abstract: Since it is unknown for many applications, which PFASs are used and how they enter the environment, target analysis-based methods reach their limits. The two most frequently used sum parameters are the adsorbable organically bound fluorine (AOF) and the extractable organically bound fluorine (EOF). Both can be quantified using either combustion ion chromatography (CIC) or high resolution-continuum source-graphite furnace molecular absorption spectrometry (HR-CS-GFMAS). Here we provide an insight on the advantageous and disadvantageous of both sum parameters and both detection methods. Our study is based on the analysis of surface water samples. Next to total fluorine (TF) analysis, AOF and EOF were determined as well as CIC and HR-CS-GFMAS are compared and results are comparatively discussed. Fluorine mass balancing revealed that, the AOF/TF proportion was higher than the EOF/TF proportion. The AOF made up 0.14–0.81% of TF and the EOF 0.04–0.28% of TF. Although, organically bound fluorine represents only a small portion of TF, PFASs are of worldwide concern, because of their extreme persistence and their bioaccumulation potential. The EOF-HR-CS-GFMAS method turned out to be more precise and sensitive than the AOF-CIC method and is a promising tool for future monitoring studies/routine analysis of PFASs in the environment.
Biocides used as disinfectants are important to prevent the transmission of pathogens, especially during the current antibiotic resistance crisis. This crisis is exacerbated by phenotypically tolerant persister subpopulations which can survive transient antibiotic treatment and facilitate resistance evolution. Despite the transient nature of disinfection, knowledge concerning persistence to disinfectants and its link to resistance evolution is currently lacking. Here, we show that E. coli displays persistence against a widely used disinfectant benzalkonium chloride (BAC). Periodic, persister-mediated failure of disinfection rapidly selects for BAC tolerance. BAC tolerance is associated with reduced cell surface charge and mutations in the novel tolerance locus lpxM. Moreover, the fitness cost incurred by BAC tolerance turned into a fitness benefit in the presence of antibiotics, suggesting a selective advantage of BAC-tolerant mutants in antibiotic environments. Our findings provide a mechanistic underpinning for the faithful application of disinfectants to prevent multi-drug-resistance evolution and to steward the efficacy of biocides and antibiotics.