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Virologists. You might know a couple of them, but unless you are a virologist yourself, the probability that you have collaborated with one in the past is low. The community is relatively small, but they pack a heavy punch and are expected to play a leading role in the research into pathogens that lies ahead. You may ask why we think virologists are our future. Suffice it to say that it is not just because they have invented technologies that belong to the space age, including use of viruses as vehicles to shuttle genes into cells[1], organic nanoparticles with specific tools attached to their surfaces to get inside target cells[2], and using genetically modified viruses as therapies to fight against cancer[3]. Did you know that virologists currently only know of about 3,200 viral species but that more than 320,000 mammal-associated viruses[4] are thought to await discovery? Just think about the viruses hidden in the Arctic ice[5] or in the insects and other animals from once cut-off regions in the world, which now face ever-increasing human exposure[6]. But a heroic (as well as an apocalyptic) role for virologists may also be on the horizon, as the adoption of phage therapy may, in the future, be used to control harmful bacteria when antibiotics fail
Soiling of photovoltaic (PV) systems compromises their performance causing a significant power loss and demanding periodical cleaning actions. This phenomenon raises great concerns in the solar energy field, thus leading to notable research efforts over the last decades. Soiling is caused by a dual action of dust deposition and biofouling. However, surprisingly, the microbiological contribution to PV soiling is often overlooked or underestimated. In this study, a variety of qPCR-based methods have been developed to quantify the microbial load of fungi, bacteria and phototrophs on PV panels. These protocols were evaluated by comparison with culturedependent methods, and were implemented with real solar plants for two years. The results show that the developed molecular methods are highly sensitive and reliable to monitor the microbial component of the soiling. Fungal biomass was clearly dominant in all analysed PV modules, while bacteria and phototrophs showed much lower abundance. Light microscopy and qPCR results revealed that melanised microcolonial fungi and phototrophs are the main biofilm-forming microorganisms on the studied solar panels. In particular, the fungal qPCR protocol is proposed as a useful tool for monitoring of PV soiling, and investigating the microbial contribution to specific soiling cases.
Recent DNA-based studies have shown that the built environment is surprisingly rich in fungi. These indoor fungi – whether transient visitors or more persistent residents – may hold clues to the rising levels of human allergies and other medical and building-related health problems observed globally. The taxo¬nomic identity of these fungi is crucial in such pursuits. Molecular identification of the built mycobiome is no trivial undertaking, however, given the large number of unidentified, misidentified, and technically compromised fungal sequences in public sequence databases. In addition, the sequence metadata required to make informed taxonomic decisions – such as country and host/substrate of collection – are often lacking even from reference and ex-type sequences. Here we report on a taxonomic annotation workshop (April 10–11, 2017) organized at the James Hutton Institute/University of Aberdeen (UK) to facilitate reproducible studies of the built mycobiome. The 32 participants went through public fungal ITS bar¬code sequences related to the built mycobiome for taxonomic and nomenclatural correctness, technical quality, and metadata availability. A total of 19,508 changes – including 4,783 name changes, 14,121 metadata annotations, and the removal of 99 technically compromised sequences – were implemented in the UNITE database for molecular identification of fungi (https://unite.ut.ee/) and shared with a range of other databases and downstream resources. Among the genera that saw the largest number of changes were Penicillium, Talaromyces, Cladosporium, Acremonium, and Alternaria, all of them of significant importance in both culture-based and culture-independent surveys of the built environment.
Tools for the characterization of secondary raw materials - status of leaching test application
(2018)
Leaching tests are used as tools for the assessment of environmental compatibility of granular waste materials with potential for reuse. The presentation gives an overview on available leaching tests, their field of application and relation to legislation. Furthermore robustness testing to elaborate optimized test conditions as well as performance characteristics from validation studies by BAM are addressed.
Tools for the characterization of secondary raw materials - status of leaching test application
(2018)
Leaching tests are used as tools for the assessment of environmental compatibility of granular waste materials with potential for reuse. The presentation gives an overview on available leaching tests, their field of application and relation to legislation. Furthermore robustness testing to elaborate optimized test conditions as well as performance characteristics from validation studies by BAM are addressed.
Development of a methodical approach for in-situ analysis of modern reverse paintings on glass
(2018)
The technique of painting on the reverse side of glass was rediscovered by artists in the early 20th century and gained great popularity, especially in Germany. In contrast to other paint techniques (e.g. panel and mural painting), the paint layers are applied in reverse succession starting with the foremost paint layer and ending with the primer (backmost layer). The paintings are viewed in reflected light, thus revealing an impressive gloss, luminosity and depth of colour.
Scientific investigation of the material provides important information for appropriate conservation concepts. Transport of the precious and fragile objects to the lab is often not feasible. Therefore, in-situ, non-invasive analysis is necessary to analyse colorants and binders. However, some analytical problems need to be considered: due to the reverse paint stratigraphy in reverse paintings on glass, the measured layer is always the backmost one. The analytical possibilities are extremely reduced, when the back is covered by a metal foil (or by paper, carton etc.). However, measurements through the glass (using Raman and VIS) can still yield information on the colourants of the front layer.
When the paint layer is accessible, we start our procedure using X-ray fluorescence (Tracer III-SD, Bruker AXS Microanalysis GmbH, 40 kV, 15 μA) and VIS reflectance spectroscopy (SPM 100, Gretag-Imaging AG). Both techniques provide first information on the inorganic colourants. Raman measurements (i-Raman®Plus, Bwtek inc., 785 nm, 50× objective, resolution 4 cm-1) are then carried out to clarify uncertain XRF measurements and to identify synthetic organic pigments (SOP). Diffuse Reflection-Infrared-Fourier-Transform Spectroscopy (DRIFTS; ExoScan, Agilent GmbH, 4000–650 cm-1, 500 scans, spectral resolution 4 cm-1) is used for the classification of the binders. Moreover, it may help to identify SOP, when fluorescence dominates the Raman signal. This analytical sequence yields the best results, when time is the limiting factor.
We present two in-situ studies of the paintings “Kreuzabnahme” (1914-15) by Carlo Mense and “Stadt am Morgen” (1921) by Walter Dexel. The painting “Kreuzabnahme” is an outstanding piece in Mense’s ɶuvre, because the glass plate was painted on both sides (Fig. 1). The results of the back-side show mainly inorganic pigments: basic lead white, talc, red and brown ochre, cinnabar, chrome yellow, viridian, ultramarine blue, Prussian blue and bone black. The classification of binding media using DRIFTS yields positive results for drying oil. Acrylic resin could be identified in two areas, resulting from a previous restauration treatment. For the front side painting, Mense used basic lead white, cinnabar, chrome yellow and umbra as pigments and oil as binder.
Measurements of the abstract painting “Stadt am Morgen” by Walter Dexel show zinc white, basic lead white, chalk, cinnabar, red lead, strontium yellow, cadmium yellow, cobalt blue, Prussian blue, ultramarine blue, brown ochre and bone black as pigments. Moreover, synthetic alizarin (PR83) was identified as dark red colorant. The results of DRIFTS classify oil as binding media.
We conclude that, the use of complementary spectroscopic methods yields the best results for in-situ analysis of reverse paintings on glass.
Zearalenone (ZEN) and its sulfate and glucoside conjugates have been detected in (a broad variety of) food and feed commodities1. Both conjugated derivatives are formed as part of fungal or plant secondary metabolism and thus, belong to the group of modified mycotoxins2. After consumption of contaminated foodstuff, the conjugates can be hydrolyzed by human intestinal microbiota leading to liberation of ZEN that implies an underestimation of the true ZEN exposure. In order to include ZEN conjugates in routine analysis, as well as for toxicological investigation reliable standards are needed.
The objective of the present study was to develop a simple and economic method for biosynthesis of ZEN conjugates. Preceding experiments on the biotransformation of ZEN by Rhizopus and Aspergillus species showed a mixed metabolite formation3. Therefore, these known ZEN conjugating fungal strains were screened for their potential to selectively synthesize the ZEN derivatives ZEN-14-sulfate (Z14S), ZEN-14-glucoside (Z14G) and ZEN-16-glucoside (Z16G). The screening was conducted by adding ZEN to liquid fungal cultures. Cultivation conditions and ZEN incubation time were varied. All media samples were analyzed for metabolite formation by HPLC-MS/MS. Z14S was exclusively formed by A. oryzae. Under optimized conditions a specific biosynthesis of Z14G by R. oryzae and Z16G by R. oligosporus was achieved. After liquid-liquid-extraction and preparative chromatographic cleanup 1H-NMR purities of ≥ 73% for Z14S, ≥ 82% for Z14G and ≥ 50% for Z16G were obtained. In addition, a consecutive biosynthesis was developed by first using Fusarium graminearum for ZEN biosynthesis on rice based liquid medium. After inactivation of Fusarium the subsequent conjugation reaction was conducted utilizing Aspergillus and Rhizopus species under the various optimized conditions.
In this study an easy and cost-efficient biosynthesis for Z14S, Z14G and Z16G was developed. The developed biosynthesis could be also used for other metabolites like ZEL conjugates. Our results of the in vitro screening indicate also the formation of a ZEL-glucoside and α ZEL-sulfate as major metabolites by R. oryzae. In sum, under optimized cultivation conditions fungi can be easily utilized for a targeted and stereospecific synthesis of ZEN conjugates.
Populations of genetically identical cells can display marked variation in phenotypic traits; such variation is termed phenotypic heterogeneity. Here, we investigate the effect of substrate and electron donor limitation on phenotypic heterogeneity in N2 and CO2 fixation in the green sulphur bacterium Chlorobium phaeobacteroides. We grew populations in chemostats and batch cultures and used stable isotope labelling combined with nanometer‐scale secondary ion mass spectrometry (NanoSIMS) to quantify phenotypic heterogeneity. Experiments in H2S (i.e. electron donor) limited chemostats show that varying levels of NH4+ limitation induce heterogeneity in N2 fixation. Comparison of phenotypic heterogeneity between chemostats and batch (unlimited for H2S) populations indicates that electron donor limitation drives heterogeneity in N2 and CO2 fixation. Our results demonstrate that phenotypic heterogeneity in a certain metabolic activity can be driven by different modes of limitation and that heterogeneity can emerge in different metabolic processes upon the same mode of limitation. In conclusion, our data suggest that limitation is a general driver of phenotypic heterogeneity in microbial populations.
Most microorganisms live in environments where nutrients are limited and fluctuate over time. Cells respond to nutrient fluctuations by sensing and adapting their physiological state. Recent studies suggest phenotypic heterogeneity in isogenic populations as an alternative strategy in fluctuating environments, where a subpopulation of cells express a function that allows growth under conditions that might arise in the future. It is unknown how environmental factors such as nutrient limitation shape phenotypic heterogeneity in metabolism and whether this allows cells to respond to nutrient fluctuations. Here, we show that substrate limitation increases phenotypic heterogeneity in metabolism, and this heterogeneity allows cells to cope with substrate fluctuations. We subjected the N2-fixing bacterium Klebsiella oxytoca to different levels of substrate limitation and substrate shifts, and obtained time-resolved single-cell measurements of metabolic activities using nanometre-scale secondary ion mass spectrometry (NanoSIMS). We found that the level of NH4+ limitation shapes phenotypic heterogeneity in N2 fixation. In turn, the N2 fixation rate of single cells during NH4+ limitation correlates positively with their growth rate after a shift to NH4+ depletion, experimentally demonstrating the benefit of heterogeneity. The results indicate that phenotypic heterogeneity is a general solution to two important ecological challenges - nutrient limitation and fluctuations - that many microorganisms face. Currently, we use NanoSIMS to develop a new approach that defines functionally-relevant, phenotypic biodiversity in microbial systems. In the last part of my presentation, I will highlight why the concept of phenotypic diversity is relevant for the understanding of antimicrobial resistance.
Surface characterisation of biofilms by nearambient pressure X-ray photoelectron spectroscopy
(2018)
The XPS information depth of approximately 10 nm is in the same size range as the outer membrane of the gram-negative bacteria, which makes XPS a suitable tool for determining the elemental composition of the bacterial surface and monitor changes caused by outer stress like dehydration or exposure to antimicrobials. However, bacteria are inherently in a hydrated state, and therefore only compatible to ultra-high vacuum after extensive sample preparation, which may degrade the sample constituents. This issue is addressed by the development of near-ambient pressure (NAP)-XPS, which enables bacteria and biofilms to be characterised in their native wet state. Artificial biofilms, bacteria and biofilms of Escherichia coli have been characterised with the laboratory NAP-XPS instrument EnviroESCA from SPECS GmbH, at pressures ranging from high vacuum to 12 mbar, and in both humid and dry environment. By studying biological samples in their native wet state, new insight about composition and transport of drugs through cell membranes and the extracellular polymeric substance (EPS) of biofilms can be obtained.
The XPS information depth of approximately 10 nm is in the same size range as the outer membrane of the gram-negative bacteria, which makes XPS a suitable tool for determining the elemental composition of the bacterial surface and monitor changes caused by outer stress like dehydration or exposure to antimicrobials. However, bacteria are inherently in a hydrated state, and therefore only compatible to ultra-high vacuum after extensive sample preparation, which may degrade the sample constituents. This issue is addressed by the development of near-ambient pressure (NAP)-XPS, which enables bacteria and biofilms to be characterised in their native wet state. Artificial biofilms, bacteria and biofilms of Escherichia coli have been characterised with the laboratory NAP-XPS instrument EnviroESCA from SPECS GmbH, at pressures ranging from high vacuum to 12 mbar, and in both humid and dry environment. By studying biological samples in their native wet state, new insight about composition and transport of drugs through cell membranes and the extracellular polymeric substance (EPS) of biofilms can be obtained. In this contribution, the latest progress on biofilm characterisation by NAP-XPS will be presented, and measurement capabilities and limitations will be discussed.
This application note presents how EnviroESCA can be used to analyze bacterial samples under near ambient pressure conditions in various states of hydration using different levels of humidity. Such investigations of bacterial cell wall surfaces in their hydrated state are essential for studying biological interfaces at work.
The use of innovative near-ambient pressure (NAP-)XPS instrumentation allows the detailed analysis of irregularly-surfaced biofilms. NAP-XPS enables the surface analysis of bacterial samples in their natural hydrated state without complex sample preparation techniques such as freeze-drying or fast-freezing, which are needed for XPS analysis in ultrahigh vacuum.
Recent studies have shown that host-microbiota interactions can lead to dramatic changes in host phenotype especially behaviour. We investigate the causal drivers of microbe-associated shifts in host phenotype by examining how feeding behaviour is modulated by a bacterial infection in an omnivorous cockroach: Blatta orientalis. We conducted food-choice experiments after challenging hosts with the common entomopathogenic soil bacterium Pseudomonas entomophila to understand the impact of bacterial pathogens on host macronutrient preference. We find that immune challenge by this bacterium drives a sharp decline in carbohydrate intake and results in a relative increase in the ratio of protein to carbohydrate consumed. Additionally, infected cockroaches reduce their overall nutrient intake. We show for the first time that cockroach feeding behaviour is dynamically modulated by a pathogen. In contrast to studies on Spodoptera moths, this modulation does not impact any of the immune parameters we measured: it does not affect the abundance of immune related proteins in the hemolymph, hemolymph antimicrobial activity, or survival. This leads to the possibility that as long-lived omnivorous species B. orientalis may be better adapted to unpredictable variation in food availability and quality. An illness-induced anorexia-like response which is thought to assist hosts in limiting nutritional resources available to pathogens is therefore the most likely cause of the observed phenotype. Reduction of carbohydrate intake by sick individuals would be consistent with such an explanation.
In another cockroach species Blatella germanica we also investigate whether in addition to pathogens, host microbiota especially gut commensals are causally responsible for shifts in host behavioural phenotype and immune competence. To address this, we conduct food-choice and immunechallenge experiments in germ-free cockroaches and naïve cockroaches.
In the near future, phosphorus (P) recycling will gain importance in terms of decreasing primary resources. Sewage sludge (SSL) is an adequate secondary P-resource for P-fertilizer production but it is also a sink for heavy metals and organic pollutants. The present study is an investigation on thermochemical P-recycling of SSL. Various temperatures and amendments were tested regarding their performance to remove heavy metals and polycyclic aromatic hydrocarbons (PAH) and simultaneous increase of the plant-availability of P. The investigations were carried out on two types of SSL originating from wastewater treatment plants with chemical P-precipitation and enhanced biological P-removal, respectively. The results show that thermochemical treatment with chlorine donors is suitable to remove the majority of heavy metals and that a combination of a gaseous chlorine donor (HCl) and sodium additives leads to both high heavy metal removal and high plant availability of P. Furthermore, plant experiments Show that almost all investigated thermochemical treatments can significantly reduce the bioavailability and plant uptake of heavy metals. Furthermore, PAHs are secondarily formed during low-temperature treatments (400–500 ° ), but can be significantly reduced by using sodium carbonate as an additive.
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).
Surface characterisation of Escherichia coli under various conditions by near-ambient pressure XPS
(2018)
Bacteria are inherently in a hydrated state and therefore not compatible to ultra-high vacuum techniques such as XPS without prior sample preparation involving freeze drying or fast freezing.
This has changed with the development of near-ambient pressure (NAP)-XPS, which makes it possible to characterise the bacterial surface with minimal sample preparation. This paper presents NAP-XPS measurements of Escherichia coli under various NAP conditions: at 11 mbar in a humid environment, at 2 mbar after drying in the chamber, pre-dried at 4 mbar, and at 1 mbar after overnight pumping at 10^−4 mbar. The high-resolution spectra of carbon, nitrogen, and oxygen are presented and found to be in general agreement with XPS measurements from freeze-dried and fast-frozen bacteria. However, it was found that the amount of carbon components associated with polysaccharides increases relative to aliphatic carbon during drying and increases further after overnight pumping. This implies that drying has an impact on the bacterial surface.
Z02 is one of the three technically supporting projects at the CSMC. In collaboration with Z01 and Z03, it aims at bridging the gap between humanities and natural sciences and technology. To that purpose, we set up a laboratory with a range of high-end instruments, most of them mobile, allowing thorough non-destructive analysis of manuscripts. In addition to working on constantly improving the laboratory and the methods of analysis, a substantial part of our activities is dedicated to service, by supporting different research projects conducted at the center.
In this talk, we will present our equipment and the possibilities offered by the different techniques available regarding the different kinds of missions: typology and classification of inks, provenance studies, recovery of faded inscriptions and palimpsests, reconstruction of the history of manuscripts, authentication and dating. We will give a brief overview of our past and ongoing activities in the frame of the second phase of the CSMC. Finally, a selection of three projects will be presented in greater detail to highlight the possibilities of our laboratory and the diversity of missions which can be carried out:
- Support of project C08 on East Frankish manuscripts containing collections of formulae
- Identifying color materials applied in Muhammad Juki's Shahnamah with non-invasive combined methods
- Checking for the presence of metals in the Herculaneum papyri
A presentation held for the seminar "Ausgewählte analytische Methoden der Physik" hosted by Prof. Birgit Kanngießer at TU Berlin. The first part focus on depth-dependent XPS-measurements (XPS, synchrotron HAXPES) to obtain a concentration profile of iodine in an artificial biofilm. In the second part, NAP-XPS measurements of various bacterial samples are presented.
Subaerial biofilms (SAB) are an important factor in weathering, biofouling, and biodeterioration of bare rocks, building materials, and solar panel surfaces. The realm of SAB is continually widened by modern materials, and the settlers on these exposed solid surfaces always include melanized, stress-tolerant microcolonial ascomycetes. After their first discovery on desert rock surfaces, these melanized chaetothyrialean and dothidealean ascomycetes have been found on Mediterranean monuments after biocidal treatments, Antarctic rocks and solar panels. New man-made modifications of surfaces (e.g., treatment with biocides or photocatalytically active layers) accommodate the exceptional stress-tolerance of microcolonial fungi and thus further select for this well-protected ecological group. Melanized fungal strains were isolated from a microbial community that developed on highly photocatalytic roof tiles after a long-term environmental exposure in a maritime-influenced region in northwestern Germany. Four of the isolated strains are described here as a novel species, Constantinomyces oldenburgensis, based on multilocus ITS, LSU, RPB2 gene phylogeny. Their closest relative is a still-unnamed rock-inhabiting strain TRN431, here described as C. patonensis. Both species cluster in Capnodiales, among typical melanized microcolonial rock fungi from different stress habitats, including Antarctica. These novel strains flourish in hostile conditions of highly oxidizing material surfaces, and shall be used in reference procedures in material testing.