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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.
Antimicrobial resistance (AMR) is a global health problem. It is well known that antibiotics can drive evolutionary processes that underlie antimicrobial resistance (AMR) evolution and spread in clinical and environmental settings. In contrast, less is known about the effects of antimicrobial substances that are used as biocides (i.e. disinfectants and preservatives) on AMR evolution and spread. Biocides are present in various settings, interacting with diverse microbial communities. Therefore, it is crucial to evaluate their role in the evolution and dissemination of antimicrobial resistance. Biocides occur in a wide range of concentrations in various environmental settings. By examining how the various concentrations affect selection mechanisms, we gain insights into potential developments related to antimicrobial resistance. The aim of this PhD thesis is to investigate the effects of biocides on processes underlying resistance evolution. Specifically, the work focused on key mechanisms for resistance spread, resistance evolution, and the effect of selection pressures on evolved resistance mechanisms. The thesis is structured around three major objectives: (i) to determine the effect of biocides on the evolution of resistance by affecting the rate of occurrence of de novo mutations, (ii) to determine the effect of biocides on the spread of resistance genes by modifying the rate of horizontal gene transfer (HGT) processes, and (iii) to investigate the selective drivers of the emergence of antimicrobial resistance in adaptive laboratory evolution (ALE) experiments. De-novo mutations are spontaneous mutations that occur at a certain rate in microorganisms. The effect of biocides at subinhibitory environmentally relevant concentrations on the mutation rate in Acinetobacer baylyi, Bacillus subtilis and Escherichia coli was assessed with the fluctuation assay. The results showed that biocides affected mutation rates in a species and substance dependent matter. The bisbiguanide chlorhexidine digluconate, the quaternary ammonium compound didecyldimethylammonium chloride, the metal copper, the pyrethroid-insecticide permethrin, and the azole-fungicide propiconazole increase mutation rates in E. coli, whereas no increases were identified for B. subtilis and A. baylyi. Horizontal gene transfer refers to diverse mechanisms that mediate the transfer of mobile genetic elements between microorganisms. This work focused on conjugation and transformation. Conjugation is a process whereby a conjugative plasmid is transferred from a donor cell to a recipient cell. Transformation is a process whereby exogenous donor DNA is taken up into a recipient cell and integrated into the recipient’s’ genome. The effects of subinhibitory environmentally relevant biocide concentrations on the conjugation rate of E. coli and the transformation rate of the naturally competent organisms A. baylyi in were assessed. The results showed that benzalkonium chloride (BAC), chlorhexidine and permethrin increased conjugation in E. coli, while none of the biocides increased transformation rates in A. baylyi. To further understand the molecular mechanisms underlying the effects on mutation and conjugation rates, I investigated the induction of the RpoS-mediated general stress and the RecA-linked SOS response upon biocide exposure. The results show a link between the general stress and the SOS response with increased rates of mutation and conjugation, but not for all biocides. One major approach to study the evolutionary response of bacteria to antimicrobials are ALE experiments with growth at subinhibitory concentrations linked to serial subculturing over many generations. Such experiments have been used to study resistance evolution to antibiotics and biocides. However, previous work showed that adaptation to biocide stress may be mediated by different evolutionary drivers. Here, I investigated the contributions of evolution for increased survival as opposed to improved growth in ALE experiments with E. coli exposed to subinhibitory BAC concentrations. Two distinct evolutionary treatments selecting for survival only or survival and growth led to specific evolutionary adaptations apparent in the phenotypes and genotypes of the evolved populations. Populations growing in the presence of BAC evolved increased fitness in the presence of BAC associated with higher resistance to BAC and cross-resistance to antibiotics, while this was not the case for populations evolving for increased survival only. Genotypic characterization by whole genome sequencing of the evolved populations revealed parallelism in mutated genes among replicate populations and distinct differences across treatments. Treatments selecting for survival and growth showed mutations in stress response related genes (hslO and tufA), while selection for survival led to mutations in genes for metabolic regulation (cyaA) and cellular structure (flagella fliJ). In summary, this thesis shows that biocides affect AMR evolution and emphasizes the importance of understanding of how biocides impact the molecular and evolutionary process that underlie AMR evolution.
Advanced light weight applications like aircrafts and wind turbine blades are made of fibre reinforced plastics (FRP) with continuous fibre reinforcement and must withstand a high thermo-mechanical cyclic loading. The quality of the fibre matrix interface has a high impact on the fatigue life and was continuously improved over the years since the 50th. The fatigue life of glass fibre reinforced plastics (GFRP) used in aircraft industry is 10 to 100 times higher compared to glass fibre non crimp fabrics used for wind turbine blades.
To assure a constant and reliable high quality and strength of reinforcement fibres, synthetic fibre production is state of the art (CF, GF). There is a need for recycling GFRP and CFRP waste due to the upcoming use. Pyrolysis and solvolysis are more expensive than the mechanical route however enable a more sustainable recycling. Natural fibres and recycled synthetic fibres have a high scatter in quality and strength. Hence it is a challenge to optimize the production / recycling processes to get a reliable quality for any demanding (second life) application.
Chemical routes for using renewables resources and recycling, is going to be a good approach especially for polymer-matrix systems to get 100% quality (back) compared to the state of the art.
Finally, a proper design, life-time extension and repair is preferable to recycling to keep the carbon footprint as low as possible.
The research project focuses on tackling the detection, measurement, and elimination of per- and polyfluoroalkyl substances (PFAS) from polluted groundwater, with a particular emphasis on addressing short (C4-C7) and ultrashort (C1-C3) chain PFAS. Given the widespread use of PFAS in various products, they are commonly found in groundwater near industrial and military sites in Germany and Israel. Moreover, recent regulations limiting the use of long chain PFAS have led industries to shift towards shorter chain alternatives. Hence, our efforts are geared towards refining detection, quantification, and removal methods for short and ultrashort chain PFAS. In terms of detection, ww are developing passive sampling devices capable of collecting and tracking the temporal distribution of PFAS species in groundwater. This will enable us to analyze contaminations in German and Israeli groundwater using cutting-edge analytical techniques. Additionally, contaminated groundwater will undergo a two-stage treatment process aimed at concentrating the relatively low PFAS concentrations using innovative membrane technologies such as closed-circuit reverse osmosis and mixed matrix composite nanofiltration membrane adsorbers. Subsequently, the streams containing higher PFAS concentrations will be treated through coagulation, with the remaining PFAS being adsorbed onto carbonaceous nanomaterials. The outcome of this research will include the creation of advanced tools for detecting, measuring, and eliminating PFAS from polluted groundwater, while also enhancing our understanding of the scope of these contaminations.
Ultrashort PFAS (≤ 3 carbon atoms) were overlooked for a long time in analytical monitoring. Beside through the use of these substances, they contribute to the PFAS background in the environment through (environmental/ bio-) degradation and incomplete destruction3 of PFAS with longer carbon chains or other fluorinated compounds. As part of the German-Israeli Cooperation in Water Technology Research project „Detection, quantification, and treatment of per- and polyfluoroalkyl substances in groundwater“ (DEFEAT-PFAS), we are developing an as simple as possible direct headspace (HS-)GC-MS method to detect trifluoroacetic acid (TFA) and perfluoropropanioc acid (PFPrA), as well as trifluoroethanol (TFEtOH), pentafluoropropanol(PFPrOH) and hexafluoroiospropanol (HFIP) in water samples. Here we present the results of the PFAS mentioned in spiked ultrapure water solutions.
Per- and polyfluoroalkyl substances (PFAS) have been extensively utilized in various industrial processes, resulting in elevated concentrations in landfills and drinking water reservoirs. Despite recognizing that shortchained
PFAS are harmful, they are often overlooked. Short-chain PFAS are more challenging to remove via adsorption and membrane separation processes, and their detection is complex, thus creating a critical gap in understanding their environmental impact. To improve their environmental monitoring, we aim to improve short-chain PFAS adsorption and electro-sorption on novel carbon-based adsorbers such as pristine and functionalized multi-walled carbon nanotubes (MWCNTs) and assess their adsorption mechanisms. Based on the obtained result, we aim to develop a PFAS passive sampling device.
Detection, Quantification and Treatment of Per and Polyfluoroalkyl substances (PFAS) in Groundwater
(2024)
The research project focuses on tackling the detection, measurement, and elimination of per- and polyfluoroalkyl substances (PFAS) from polluted groundwater, with a particular emphasis on addressing short (C4-C7) and ultrashort (C1-C3) chain PFAS. Given the widespread use of PFAS in various products, they are commonly found in groundwater near industrial and military sites in Germany and Israel. Moreover, recent regulations limiting the use of long chain PFAS have led industries to shift towards shorter chain alternatives. Hence, our efforts are geared towards refining detection, quantification, and removal methods for short and ultrashort chain PFAS. In terms of detection, ww are developing passive sampling devices capable of collecting and tracking the temporal distribution of PFAS species in groundwater. This will enable us to analyze contaminations in German and Israeli groundwater using cutting-edge analytical techniques. Additionally, contaminated groundwater will undergo a two-stage treatment process aimed at concentrating the relatively low PFAS concentrations using innovative membrane technologies such as closed-circuit reverse osmosis and mixed matrix composite nanofiltration membrane adsorbers. Subsequently, the streams containing higher PFAS concentrations will be treated through coagulation, with the remaining PFAS being adsorbed onto carbonaceous nanomaterials. The outcome of this research will include the creation of advanced tools for detecting, measuring, and eliminating PFAS from polluted groundwater, while also enhancing our understanding of the scope of these contaminations.
In order to protect natural gypsum deposits and to compensate for the decreasing amount of Flue Gas Desulfurization (FGD) gypsum it is necessary to develop and explore new sources of gypsum. For this purpose, the potentials of different gypsum wastes are investigated in the study “GipsRec 2.0”, funded by the Federal Ministry of Education and Research (Germany).
On the one hand, the project worked on a new processing technology for gypsum fiberboards (GFB). While the recycling of gypsum plasterboards has already been carried out on an industrial scale for several years, the recycling of gypsum fiberboards (GFB) has proven to be challenging. Gypsum fiberboards from demolition sites and offcuts from GFB production were used for these investigations. The tests were conducted on a technical scale. Furthermore, various synthetic gypsums are being investigated with regard to their suitability for gypsum production. The analyses are carried out on production residues.
In this project, a promising process for gypsum fiberboard recycling could be developed, as well as other waste gypsums are investigated and evaluated with regard to their potential as secondary raw material. In addition, selected process routes are assessed for their environmental impact using a life cycle assessment (LCA) approach.
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.
Any surface in the environment acts as hotspot for microbial attachment and activity. These biofilms represent the interface between humans and the environment.
While in the past biofilms were often seen as disturbance, we now start to understand the enormous potential of beneficial biofilms. They can be used in a broad range of applications and are sources for new microorganisms and traits. After all, biofilms represent a great example for a collaborative lifestyle.