4 Material und Umwelt
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Harnessing near-chromosome level quality genomes to explore the evolution of termite immunity
(2023)
The phylogeny of the Blattodea boasts a wide degree of sociality spanning from solitary cockroaches to advanced ecosystem-dominating higher termite societies. The emergence of sociality in termites was associated with the acquisition of a diverse range of social structures. Previous work has found evidence for a caste-specific social defence system in termites leading to an immune system that may favour group over individual defence. While preliminary work suggests a correlation between social transitions and a reduction of immune gene family diversity, the lack of available high-quality termite genomes hampers complete knowledge of the true diversity of immune gene evolution across termite phylogeny. Here, we report on the sequencing and near-chromosome level assembly of 48 high-quality long-read-based genomes across major termite and cockroach sister-branch lineages. We investigate the diversity and evolutionary history of immune genes across genomes, focusing particular attention on correlations between immune gene evolution and transitions in sociality over termite phylogeny.
Insecta is one of the most diverse phyla in the animal kingdom, with species living in all types of habitats encountering an even greater diversity of pathogens and parasites. Defence strategies against such harmful threats led to a variety of molecular mechanisms, ecological shifts, and genetic innovations. Gene families underlying the molecular basis of the immune responses have evolved within the boundaries given by the species ecology. Here, we explore the evolution of some emblematic immune gene families throughout the Insecta phylum shedding light on remarkable genetic events such as gene duplication or gene loss. We followed a workflow based on the Hidden Markov model to search for immune genes in 55 high-quality genomes of insects. We highlight the emergence of group defence in social species as an ecological shift that released selection pressure on immune gene families notably in Blattodea. Further, we draw attention to certain gene families and the link between their diversity and the specificities of the species’ microbiota. Overall, we report data on immune gene diversity in insects.
The phylogeny of the Blattodea boasts a wide degree of sociality spanning from solitary cockroaches to advanced ecosystem-dominating higher termite societies. The emergence of sociality in the termites was associated with the acquisition of a diverse range of social structures and differential expression of specific gene network. Previous work has found evidence for a caste-specific social defence system in termites leading to an immune system that may favour group over individual defence. While preliminary work suggests a correlation between social transitions and a reduction of immune gene family diversity, the lack of available high-quality termite genomes hampers complete knowledge of the true diversity of immune gene evolution across termite phylogeny. Here, we report on the sequencing and assembly of 50 high-quality long-read-based genomes and 180 caste- and sex-specific brain transcriptomes across major termite and cockroach sister-branch lineage. We investigate the diversity and evolutionary history of immune genes across genomes, and the potential gene networks that have evolved with the emergence of termite sociality and some aspect of immune related behaviours.
Microbial contamination of fuels by fungi or bacteria poses risks such as corrosion and fuel system fouling, which can lead to critical problems in refineries and distribution systems and has a significant economic impact at every stage of the process. Many factors have been cited as being responsible for microbial growth, like the presence of water in the storage tanks. In fact, only 1 % water in a storage system is sufficient for the growth of microorganisms like bacteria or yeasts, as well as for the development of fungal biomass at the oil/water interface.
This work presents a rapid test for the accurate determination of genomic DNA from aqueous fuel extracts. The detection is based on the use of polystyrene-mesoporous silica core-shell particles onto which modified fluorescent molecular beacons are covalently grafted. These beacons contain in the hairpin loop a target sequence highly conserved in all bacteria, corresponding to a fragment of the 16S ribosomal RNA subunit. The designed single-stranded molecular beacon contained fluorescein as an internal indicator and a quencher in its proximity when not hybridized. Upon hybridization in presence of the target sequence, the indicator and the quencher are spatially separated, resulting in fluorescence enhancement. To perform the assay the developed particles were deposited on different glass fibre strips to obtain a portable and sensitive rapid test. The assays showed that the presence of genomic DNA extracts from bacteria down to 50–70 μg L–1 induced a fluorescence response. The optical read-out was adapted for on-site monitoring by fitting a 3D-printed case to a conventional smartphone, taking advantages of the sensitivity of the CMOS detector. Such embedded assembly enabled the detection of genomic DNA in aqueous extracts down to the mg L–1 range and represents an interesting step toward on-site monitoring of fuel contamination.
Thermal Destruction of PFAS
(2023)
Thermal treatment processes are currently the only full-scale option for thedestruction of per- and polyfluoroalkyl substances (PFAS) in large waste streams. While all organic molecules including PFAS are susceptible to thermal destruction, their decomposition rates are controlled by process variables such as temperature, reaction atmosphere, and residence time. Concerns exist about the formation of products of incomplete destruction and their emission from hazardous waste incinerators. This talk will summarize the current stateof-the-art of thermal PFAS destruction, identify research needs, and showcase future research designed to address critical knowledge gaps.
This presentation was held in an OECD Webinar introducing the newly developed and published OECD TG 125 on particle size and size distribution. The presentation is explaining the structure if the TG 125 and addresses all included methods and methodologies in a short and understandable way for the broader public. The presentation includes sections about nano-particles and nano-fibres.
Generation of Cryomyces antarcticus mutants to explore the importance of DHN melanin for survival
(2023)
Cryomyces antarcticus, a cryptoendolithic melanized fungus endemic to Antarctica (phylum Ascomycota, class Dothideomycetes incertae sedis), has demonstrated high capability to survive extreme environmental conditions like those found in space (e.g., ionizing radiation, vacuum, microgravity), thus fueling fundamental astrobiological questions like “the search for life beyond Earth”. Its extraordinary resilience has been attributed to the presence of thick, highly melanized cell walls, which may contain both DHN and DOPA melanins. To better understand the contribution of DHN melanin to the overall resilience of C. antarcticus, we decided to generate melanin-deficient mutants by genetic engineering. For this, the melanin-PKS (polyketide synthase)- encoding ortholog was identified in the C. antarcticus CBS 116301 genome and used to design primers for re-sequencing of the capks1 locus in the strain CCFEE 515. Based on the genetic toolkit developed for the black fungus Knufia petricola, we designed a strategy for mutating capks1. Protoplasts of C. antarcticus CCFEE 515 were generated and co-transformed with a circular AMA-containing plasmid for expression and in-vivo assembly of Cas9 and two capks1-specific sgRNAs and a PCR-generated donor DNA i.e., a hygromycin resistance cassette flanked by ~75-bp-long sequences homologous to the capks1 locus. Transformation of C. antarcticus is challenging because of its very slow growth and uncertain gene copy number. It took six months from obtaining enough biomass for cell wall lysis to transferring the putatively resistant transformants for genotyping, but we eventually managed to generate three independent non-melanized (whitish) Δcapks1 mutants! We are now awaiting first complemented strains as ultimate genetic transformation control. They are considered to have a wild-type-like pigmentation due to the reintroduction of the wild-type capks1 into the Δcapks1 mutant.
In recent years the BAM has been focusing on the characterization of the writing materials of the medieval Torah scrolls. The challenge imposed by the study of this specific type of manuscript led to designing and building dedicated stages to ensure the safety of the scrolls during the analysis. Furthermore, the identification of multiple corrections executed in apparently identical inks required broadening the standard two-step protocol of near infrared reflectography and X-ray fluorescence to include multispectral imaging. This work has prepared the division to participate in the large collaborative project “To roll: Materialisierte Heiligkeit (Materialised Holiness)” based at the Free University, Berlin.
The presentation will demonstrate the aims of the studies, preliminary results and the strategies adopted by the division 4.5 of the BAM.
In-vivo mutagenesis of the rock inhabitant Knufia petricola by a customized Ac/Ds transposon system
(2023)
Microcolonial black fungi ubiquitously inhabit sun-exposed natural and man-made surfaces of our planet. To promote genetic studies, CRISPR/Cas9-based genome editing was implemented in the rock-inhabiting fungus Knufia petricola (Eurotiomycetes/ Chaetothyriales). Now efficient targeted mutagenesis of K. petricola - as a representative of the polyphyletic group of black fungi - enables the elucidation of extremotolerance, oligotrophism, unusual types of cell division, mineral weathering and symbiotic interactions. Even more progress on assigning functions to yet unknown genes can be achieved by a forward genetics approach. We chose the two-component Activator/Dissociation (Ac/Ds) transposon system from maize for generating K. petricola insertional mutants by in-vivo mutagenesis. For the optimal use of this genetic tool, an inducible promoter i.e, from the metabolism-independent Tet-on system, was combined with the AcTPase-coding sequence enabling the regulatable transposition of the resistance cassette-containing Ds transposon. In total, six auxotrophic Ac/Ds starter strains containing the Ds transposon at different position of ade2, ura3 or ppt1 were generated. The cultivation of these strains with doxycycline for induction of TET::Ac and subsequent selection of cells on ADE/URA/LYS-lacking media resulted in prototrophic colonies (revertants) for most Ac/Ds strains. Amplicon sequencing of excision sites revealed characteristic footprint patterns, proving that the transposon jumped. For identifying unknown Ds re-insertions sites, the thermal asymmetric interlaced (TAIL)-PCR was successfully implemented. First identified Ds re-insertion sites suggest that the distribution pattern may depend on the excision site. Currently, transposition frequencies and genome-wide distribution of re-insertion sites are studied in different Ac/Ds starter strains to identify the best candidate for generating saturated mutant libraries. This transposon mutagenesis strategy is also interesting for studying other black fungi, because once the Ac and Ds components are integrated in the genome, the fungus ‘only’ needs to be cultivated for generating insertional mutants.
Melanins are organic pigments produced by most fungi. These organisms either fix these pigments in their cell wall or secrete them into their extracellular environment to protect themselves against an array of physicochemical stresses (e.g., UV irradiation, desiccation, ...). Melanin can adsorb metals like Fe. How this affects fungal uptake of Fe and deterioration of Fe-containing minerals and metals is however less known. To study this, we use the model fungi Knufia petricola A95, a rock-inhabiting fungus known to deteriorate minerals and have melanised cell walls, and Amorphotheca resinae, able to contaminate fuel tanks, secrete melanin and corrode metals. In K. petricola, we have deleted genes involved in melanin production and Fe uptake using CRISPR/Cas. Through comparison of the geochemical signatures of these gene deletion mutants with those of the wild type (WT), we explore the specific mineral/metal deterioration mechanisms of melanised fungi.
Fe isotope signatures of the biomass of melanin- and Fe uptake-deficient mutants of K. petricola revealed that Fe adsorbed either directly onto melanin or after being reduced by Fe reductases. Importantly, once adsorbed to melanin, Fe could not be mobilised and taken up into the cell: both the WT and its melanin-deficient mutant, previously grown at Fe replete conditions, showed similar growth at Fe deficient conditions.
Olivine dissolution experiments revealed that Fe oxidation inhibits dissolution. K. petricola was able to enhance dissolution when this inhibition is strongest (at pH 6) and prevented dissolution when this inhibition is weakest (at pH 4). The fungus therefore dissolves olivine by interacting with the oxidised Fe at the olivine surface. However, Fe uptake did not seem to be involved: mutants deficient in various Fe uptake mechanism dissolved olivine at the same rate as the WT. This indicates that Fe adsorption onto melanin might play a key role. This is also shown by K. petricola’s ability to enhance olivine dissolution even further if secreting a melanin precursor and A. resinae’s corrosion of carbon steel whilst secreting melanin. Combined, our results imply that the Fe adsorbed to melanin cannot be taken up but enables fungi to deteriorate Fe-containing substrates at a higher rate.
Human-made systems, also called “build environment” or “technosphere”, sustain human comfort as well as our industrial activities. These systems have become particularly widespread since the Industrial Revolution, i.e., since the 17th century. At the same time, these technical systems – buildings, monuments, energy production, transformation and transmission, water purification and supply systems - serve as new habitats for living organisms.
Life is ubiquitously present on our planet since a very long time: the Earth is 4.54 billion years old and microbial communities have played a key role on our planet for 3.7 billion years. Once human-made system appeared, microorganisms became an integral part of all types of technosphere infrastructure as well.
Here we will illustrate biosphere-technosphere interactions using a specific example of the black fungi and their impact on the efficiency of solar (photovoltaic) panels. This expanding renewable infrastructure for electricity generation is growing on all continents - and create a specific, arid habitat for stress-tolerant black fungi.
Black fungi were once discovered in hot and cold natural deserts – and now belong to the persistent colonisers of human-made deserts of solar parks. This new niche is evolving an impressive biodiversity. So far more than 60 isolates of black fungi belonging to Arthoniomycetes, Eurotiomycetes and Dothideomycetes were obtained from solar panels in Europe and Americas. Here we will present the analysis of this emerging anthropogenic biodiversity.
Opportunities for future research in the field include quantification of the microbial load on technosphere surfaces – along with characterisation of the corresponding microbial diversity. The strategy of precise measurement and characterisation will enable us to reliably determine the beneficial and harmful functions that living microorganisms play in the functioning of energy-generating systems – and technosphere in general.
Volatile organic compounds (VOCs) emitted by furniture and
building materials can cause health issues. For an
improvement of indoor air quality low emitting materials
should be used. Quality assurance and –control (QA/QC)
measures require an emission reference material (ERM) with a
predictable emission rate of VOCs. The idea is to use porous
materials as ERM, which store the VOCs inside their pores and
emit them constantly.
Regional standardisation activities and how VAMAS can help in any way to promote activities are reported.
Activities related to organisational updates, government initiatives/priorities (especially related to Materials), details of any strategy documents publicly available, networks within Germany and how we engage are presented.
The overarching goal of this project is to develop a predictive model for efflux-mediated antimicrobial tolerance in bacterial multicellular assemblies. Our central hypostasis is that efflux pump activity causes emergent antibiotic tolerance of multicellular bacterial populations, through the interplay of efflux mediated spatial interactions and efflux-linked persistence. To test this hypothesis, we will use a combination of microscopy, microbial killing assays, computational modelling, and data analysis, integrating information from 3 types of multicellular assembly: colonies, cell-to-cell interactions in a monolayer microfluidic device, and 3D flow chamber biofilms. Building on our preliminary observations, we will experimentally characterize the link between colony structure and spatial patterns of efflux gene expression in strains that differ in their levels of efflux. We will develop a mathematical model to test whether local growth inhibition of neighbors due to effluxing cells, coupled with local environment-dependent regulation of efflux, can account qualitatively for these results. By including persister cell formation in our model we will predict, and measure, the emergent function of antimicrobial tolerance in our colonies. To fully understand how tolerance emerges from the interplay between efflux-mediated spatial interactions and efflux-linked persister cell formation, we need quantitative measurements at the single cell level. To this end, we will use a microfluidic setup with cells growing in a monolayer to qualify in detail the dependence of efflux expression and persister cell formation on nutrient conditions, the correlation between efflux and persister formation, and the spatial range of efflux-mediated neighbour growth inhibition. To predict and quantitatively understand the emergent multicellular function of tolerance, we will perform individual-based modelling of biofilm growth, using as input the parameters measured on the single-cell level with our microfluidics experiments. Our simulations will predict biofilm spatial structure development, patterns of efflux and persister formation and, ultimately, tolerance to antimicrobial challenge. These predictions will be directly tested in flow-cell biofilm experiments. We are currently generating acrAB-tolC knockout-strain, without efflux activity, and a strain with an inducible acrAB-tolC efflux pump. To distinguish the different strains under the microscope, they were labeled with genes encoding for different fluorescent proteins. All strains are currently characterized in terms of growth, minimum inhibitory concentration of different antimicrobial substances, colony morphology, and biofilm formation ability. On the theoretical side, we are currently working on modeling the system at various scales and degree of detail, ranging from coarse-grained continuum models to stochastic, individual-based models. Some exploratory work was doe to test existing software for individual-based modelling that may be adapted for our purpose. Furthermore, we are in the process of developing more coarse-grained models. This work involves some physiological modelling and literature search, focusing on working mechanisms of efflux pumps and kinetic models for import and export of antibiotics.
Adipokinetic hormone (AKH) is a neuropeptide produced in the insect corpora cardiaca that plays an essential role in mobilizing carbohydrates and lipids from the fat body to the hemolymph. AKH acts by binding to a rhodopsin-like G protein-coupled receptor (GPCR), the adipokinetic hormone receptor (AKHR). In this study, we tackle AKH ligand and receptor gene evolution as well as the evolutionary origins of AKH gene paralogues from the order Blattodea (termites and cockroaches). Phylogenetic analyses of AKH precursor sequences point to an ancient AKH gene duplication event in the common ancestor of Blaberoidea, yielding a new group of putative decapeptides. In total, 16 different AKH peptides from 90 species were obtained. Two octapeptides and seven putatively novel decapeptides are predicted for the first time. AKH receptor (AKHRs) sequences from 18 species, spanning solitary cockroaches and subsocial woodroaches as well as lower and higher termites, were subsequently acquired using classical molecular methods and in silico approaches employing transcriptomic data. Aligned AKHR open reading frames revealed 7 highly conserved transmembrane regions, a typical arrangement for GPCRs. Phylogenetic analyses based on AKHR sequences support accepted relationships among termite and cockroach lineages to a large extent, while putative post-translational modification sites do not greatly differ between solitary and subsocial roaches and social termites. This study provides important information not only for AKH and AKHR functional research but also for further analyses interested in their development as potential candidates for biorational pest control agents against invasive termites and cockroaches.
Consequences of tolerance to disinfectants on the evolution of antibiotic resistance in E. coli
(2023)
Biocides are used as disinfectants and preservatives; one important active substance in biocides is benzalkonium chloride (BAC). BAC-tolerant bacterial strains can survive short treatments with high concentrations of BAC. BAC tolerance and resistance have been linked to antibiotic resistance. Here, the selection dynamics between a BAC-tolerant Escherichia coli strain and a sensitive wild type were investigated under four conditions: in the absence of antibiotics and in the presence of three different sub-inhibitory concentrations of the antibiotic ciprofloxacin in liquid cultures. The wild type was selected over the BAC-tolerant strain in the absence of antibiotics, while the BAC-tolerant strain was selected over the wild type at all ciprofloxacin concentrations investigated, with a minimum selection concentration (MSC) of 1/10th of the minimum inhibitory concentration (MIC) of the wild type. Furthermore, the evolvability of resistance of the two strains to inhibitory concentrations of ciprofloxacin was assessed by performing a serial dilution evolution experiment with gradually increasing ciprofloxacin concentrations. The wild type had a higher probability to develop resistance
to ciprofloxacin than the tolerant strain. By the end of the evolution experiment both strains evolved to grow at the highest ciprofloxacin concentration investigated, which was 2048 ×MIC of the wild type. The importance of these results is highlighted by the fact that concentrations of ciprofloxacin well above the calculated MSC can be found in environmental samples such as hospital wastewaters and livestock slurry. In turn, BAC is used as a disinfectant in the same settings. Thus, the selection of BAC-tolerant strains at sub-inhibitory concentrations of ciprofloxacin can contribute to the stabilization and spread of BAC-tolerance in natural populations. The prevalence of such strains can impair the effects of BAC disinfections.
The increasing use of carbon fiber reinforced polymers (CFRP), as lightweight materials essential for the transformation, demands for a safe treatment option of carbon fiber (CF) containing waste streams. Because of their low reactivity towards oxidation processes, CF can neither be treated in a conventional nor hazardous waste incineration plant. Even the extremely high temperature in a cement rotary kiln does not lead to full conversion. To increase the sustainability of CF, other processes need to be investigated. Opening other reaction pathways, using CF waste streams as secondary resource in pyrometallurgy to replace fossil carbon, can be a solution for a safe and sustainable treatment. After first small scale experiments, CF containing waste streams were used as reductant in a semi-industrial scale electric arc furnace (EAF). First results and impressions are presented.
The increasing use of carbon fiber reinforced polymers (CFRP), as lightweight materials essential for the transformation, demands for a safe treatment option of carbon fiber (CF) containing waste streams. Because of their low reactivity towards oxidation processes, CF can neither be treated in a conventional nor hazardous waste incineration plant. Even the extremely high temperature in a cement rotary kiln does not lead to full conversion. To increase the sustainability of CF, other processes need to be investigated. Opening other reaction pathways, using CF waste streams as secondary resource in pyrometallurgy to replace fossil carbon, can be a solution for a safe and sustainable treatment. To support the industrial application, experiments on different scales were conducted. Here, results of thermochemical investigations, reactivity studies, crucible experiments and pilot plat trials are reported.
The ink analysis protocol developed through cooperation between the Bundesanstalt für Materialforschung und -prüfung in Berlin and the Centre for the Study of Manuscript Cultures at the University of Hamburg involves the use of imaging techniques for ink screening, followed by spectroscopic analysis. In our presentation, we will begin by briefly reviewing the history of writing inks and discussing the three main categories: carbon-based inks, plant, and iron-gall inks. We will address their chronology, precursors, and mixed forms, as well as the features that allow for their identification. Then, we will present the techniques we use in ink analysis, whereby we would like to highlight the limitations, advantages, and disadvantages of each approach. Finally, we will discuss the new mass-spectrometric method based on micro-sampling and using atmospheric solid analysis probe (ASAP).