4 Material und Umwelt
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- 4 Material und Umwelt (30)
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A Genetic Toolbox for Exploring the Life Style of the Rock-inhabiting Black Fungus Knufia petricola
(2019)
After their discovery on rock surfaces in cold and hot deserts, a polyphyletic group of ascomycetous black fungi was found to dominate a range of hostile environments – natural and man-made, from salterns to dishwashers, roofs and solar panels. Together with bacteria and algae they may establish sub-aerial biofilms and cause weathering of the surfaces they grow on. Their impressive survival abilities as well as their constitutive protective pigmentation and cluster-like microcolony organisation are similar in environmental isolates as well as in heat-tolerant opportunistic pathogens of animals and humans. The exact genetic properties that ensure their survival in extreme environments can be studied if some black fungi were amenable to genetic manipulations. We selected the rock-inhabiting fungus Knufia petricola (class Eurotiomycetes, order Chaetothyriales) that grows moderately in axenic culture and exhibits all the characteristics of black yeasts such as yeast-like cell growth, absence of reproductive structures and constitutive dihydroxynaphthalene (DHN) melanogenesis. For this environmental strain we developed protocols to efficiently generate and transform protoplasts resulting in stable homokaryotic transformants by targeting genes involved in pigment synthesis and expressing fluorescent reporter genes. Hence, endogenous and foreign genes can be expressed from episomal AMA1-containing plasmids and genome-integrated DNA constructs. Moderate rates of homologous recombination allow for both ectopic and targeted integrations. CRISPR-Cas9 was further validated as a strategy for obtaining selection marker-free mutants and silencing via RNA interference as an approach to study essential genes. Availability of this genetic toolbox and an annotated genome sequence is paving the way for studying interactions of K. petricola and other black yeasts with environmental stressors, material surfaces, soil matrices and phototrophic symbionts.
Black microcolonial fungi (MCF) are persistent inhabitants of rock surfaces in hostile desert environments. In these niches, MCF have evolved mineral-weathering and symbiotic capabilities as well as mechanisms to cope with multiple stresses such as solar irradiation, temperature extremes and low water activity. Due to their stress tolerance these ascomycetes are prominent in modern terrestrial ecosystems – like man-made material surfaces from roof to solar panels. MCF interactive capabilities support their facultative symbiotic relationships with cyanobacteria and ensure their rock-weathering geochemical activity.
Using the rock-inhabiting fungus K. petricola A95 (Chaetothyriales), we developed transformation protocols and deleted genes responsible for production of the protective pigments melanins and carotenoids. To confirm that the mutant phenotypes were not due to hidden mutations, melanin synthesis was restored by complementing the mutants with the respective wild type genes. Strains of K. petricola carrying gene variants for fluorescent proteins EGFP and DsRed are available. We successfully labelled the cytoplasm, nuclei, peroxisomes and mitochondria. Targeted and ectopic integrations result in stable transformants suitable for further phenotypical characterization. As K. petricola is a non-pathogenic fungus with all characteristic features of MCF, including meristematic growth, melanized cell-walls, extracellular polymeric substances and extensive pigment production, our results will shed light on protective role of pigments during cell wall maturation and oxidative stress defence in rock-inhabiting MCF. Genes involved in environmental sensing or substrate and phototroph interactions are currently targeted. With the help of a mutant collection and fluorescently labelled K. petricola we will be able to investigate interactions of MCF with environmental stressors, mineral substrates, soil matrices and phototrophic symbionts.
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.
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.
A mobile Bruker S2 Picofox TXRF spectrometer has been used in two field campaigns within the EMPIR env07 AEROMET project for the on-site analysis of cascade impactor aerosol samples.The results show that even at moderate air pollution levels – i.e.PM10 fairly below 20 μg/m³ - element mass concentrations in air in the range of 100 pg/m³could be measured in up to 13 size bins after sampling times of less than only 0.5 days.
In this poster we will present some results of a research projects dealing with Arabic recipes of black inks. To improve conservation practices of Arabic manuscripts we have assessed the feasibility of the recipes collected from many Arabic sources on bookmaking and reproduced the most common inks. Ink samples were then artificially aged and analysed using an array of analytical techniques, mostly non-invasive, in order to build a database of Arabic inks and their ingredients and also to verify the detection limits of the portable equipment employed.
First, we will provide an overview of the sources [1-2] employed in the study – dating from 9th to 14th century, although the manuscripts in which they can be found dates up to the 20th century – with an eye on the ink typologies (real and perceived by the compilers). Then we will show how, by reproducing the recipes, it was possible to shed light on some oddities in the procedures and the choice of ingredients. In the end we will discuss problems concerning the identification of ink types and ingredients using reflectography (in Vis, IR and UV), X-Ray Fluorescence, Infrared and Raman spectroscopy. The focus will be in particular on mixed inks – both in the form of carbon inks with the addition of tannins and of a mixture of carbon and iron gall inks – which are particularly difficult to detect [3]. We will also examine the results of iron gall inks made with iron filings or nails instead of vitriol and the various precursor used to extract gallic acid that are mentioned in the sources as surrogates of gall nuts.
[1] A. Schopen, Tinten und Tuschen des arabisch-islamischen Mittelalters, Göttingen: Vandenhoeck & Ruprecht, 2004
[2] S. Fani, Le arti del libro secondo le fonti arabe originali. I ricettari arabi per la fabbricazione degli inchiostri (sec. IX-XIII): loro importanza per una corretta valutazione e conservazione del patrimonio manoscritto, PhD thesis, Napoli: Università L’Orientale, 2013
[3] C. Colini et. al, Manuscript cultures, 11 (2018) 43-50
In this poster we will present some results of a research projects dealing with Arabic recipes of black inks [1-2]. To improve conservation practices of Arabic manuscripts we have assessed the feasibility of the recipes collected from many Arabic sources on bookmaking and reproduced the most common inks. Ink samples were then artificially aged and analysed using an array of analytical techniques, mostly non-invasive, in order to build a database of Arabic inks and their ingredients and also to verify the detection limits of the portable equipment employed.
We will show procedures and problems concerning the identification of ink types and ingredients using reflectography (in Vis, IR and UV), X-Ray Fluorescence, Infrared and Raman spectroscopy. The focus will be in particular on squid ink [3] and mixed inks – both in the form of carbon inks with the addition of tannins and of a mixture of carbon and iron gall inks – which are particularly difficult to detect [4]. We will also examine the results of iron gall inks made with iron filings or nails instead of vitriol, the various precursor used to extract gallic acid that are mentioned in the sources as surrogates of gall nuts.
[1] A. Schopen, Tinten und Tuschen des arabisch-islamischen Mittelalters, Göttingen: Vandenhoeck & Ruprecht, 2004
[2] S. Fani, Le arti del libro secondo le fonti arabe originali. I ricettari arabi per la fabbricazione degli inchiostri (sec. IX-XIII): loro importanza per una corretta valutazione e conservazione del patrimonio manoscritto, PhD thesis, Napoli: Università L’Orientale, 2013
[3] S. Centeno, J. Shamir Journal of Molecular Structure, 873 (2008), 149-159
[4] C. Colini et. al, Manuscript cultures, 11 (2018) 43-50
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.
This work addresses the identification of synthetic organic pigments (SOP) in ten modern reverse paintings on glass (1912-1946) by means of an in-situ multi-analytical approach. The combination of the complimentary properties of mobile Raman spectroscopy and diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) enabled the detection of sixteen SOP even in complex mixtures with inorganic compounds and binders. For the β-naphthol pigments, both Raman and DRIFTS yield appropriate results. DRIFTS was the preferred method for the detection of synthetic alizarin (PR83). Its diagnostic band pattern even allows its detection in complex mixtures with mineral pigments, binders and fillers. Raman spectroscopy yielded distinctive spectra for the triaryl carbonium pigments (PG1, PV2, PR81) and the two-yellow azo SOP (PY3, PY12), whereas DRIFT spectra were affected by extensive band overlapping. This may also occur in Raman spectra, but in less problematic amounts. Fluorescence is the major problem with Raman and it significantly hampers the SOP spectra even with the 785 nm laser. On the one hand the big spot size of DRIFTS (10 mm) limits the technique to rather large sampling areas, whereas the use of a 50× objective for in-situ Raman measurements permits a focus on small spots and aggregated SOP flakes. Moreover, “environmental” factors like temperature changes, artificial light, limited space and vibrations when people pass by need to be considered for in-situ measurements in museums.
Finally, the results show the experimental use of SOP in modern reverse glass paintings. Among several rare SOP (e.g. PB52, PR81), two of them (PG1, PV2) have never been reported before in any artwork.