TY - CONF A1 - Bell, Jérémy A1 - Climent, Estela A1 - Gotor, Raúl A1 - Tobias, Charlie A1 - Martin-Sanchez, Pedro M. A1 - Rurack, Knut T1 - Dipstick coated with polystyrene-silica core-shell particles for the detection of microbiological fuel contamination N2 - 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. T2 - IMA 2023 CY - Chania, Greece DA - 18.09.2023 KW - Teststreifen KW - Test strip KW - Microbial KW - Mikrobiell KW - Smartphone KW - Particles KW - Partikeln PY - 2023 AN - OPUS4-58526 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Catanzaro, Ilaria T1 - Generation of Cryomyces antarcticus mutants to explore the importance of DHN melanin for survival N2 - 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. T2 - IUBMB Focused Meeting on Extremophilic Fungi (FUN-EX) CY - Ljubljana, Slovenia DA - 19.09.2023 KW - Black fungus KW - Genetics KW - UV tolerance KW - Melanin PY - 2023 AN - OPUS4-58458 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Dehkohneh, Abolfazl T1 - Modelling biofilm formation and material colonisation for Knufia petricola N2 - Rock-inhabiting black fungi are adapted to survive extreme stresses such as radiation and lack of water and are thus able to colonise arid material surfaces like solar panels and marble monuments. Black fungi can typically grow as one of two cell types: depending on the environmental conditions, they are able to form yeast-like cells or hyphal filaments. Knufia petricola is one member of the rock-inhabiting black fungi which functions as a model reference species. As there is no mathematical model for biofilm formation of RIF, we are going to develop a model for K. petricola which describes fungal growth on the level of individual cells adn biofilm formation. T2 - Mini conference at University of Birmingham CY - Birmingham, UK DA - 05.10.2022 KW - Knufia petricola KW - Rock-inhabiting black fungi KW - individual-based modelling PY - 2022 AN - OPUS4-56697 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Dehkohneh, Abolfazl T1 - How Single Cells and Biofilms of the Extremotolerant Black Fungus Knufia petricola Respond to Oligotrophic Environments N2 - Melanised black fungi, such as Knufia petricola, are renowned for their constitutive melanin production, extremotolerance, and ability to thrive in hostile, oligotrophic natural and built environments from Antarctic outcrops to marble monuments and solar panels. These traits make them compelling models for astrobiological research, offering insights into survival strategies under conditions analogous to extraterrestrial habitats. Here, we quantified the responses of K. petricola A95 to carbon and nitrogen supply, varying sources (C: glucose vs. sucrose; N: ammonium vs. nitrate) and concentrations (C:N ratios of 0.6 – 600), including nutrient-free controls. Biofilm radial extension and thickness, morphology, biomass, produced CO2, and carbon-use efficiency (CUE) were analysed across controlled C:N ratios after 28 days. we combined macroscale assays of biofilm formation on agar with microscale tracking of single-cell development into microcolonies in a microfluidic device. This dual-scale approach allows the study of the nutritional requirements and growth strategies of K. petricola under chronic oligotrophy and provides quantitative inputs for individual-based simulations using iDynoMiCS 2.0. Macroscale analysis identified a C:N ratio of 60 as optimal, resulting in the highest biomass and colony thickness and the highest cumulative CO2 release. Depletion or low concentrations of C or N reduced biomass and increased peripheral filamentation and higher substrate penetration. Notably, nitrate as the N source produced biofilms with more peripheral filaments and caused more wrinkles on the surface of biofilm, whereas ammonium yielded compact, smooth biofilms with little to no filamentation. The CUE ranged from 0.4 to 0.6, being lowest under nutrient limitation. Microscale observations of single cells revealed that increasing the C:N ratio from 0.6 to 60 shortened cell cycles and promoted branching, no branching formed without C and N. Thus, a denser colony at optimal conditions and a rather extended morphology under C or N limitations were observed. T2 - Berlin Early-career Space Research (BESR) conference 2025 CY - Berlin, Germany DA - 06.10.2025 KW - Black fungi KW - Oligotrophs KW - Rock-inhabiting fungi PY - 2025 AN - OPUS4-65034 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Erdmann, Eileen T1 - In-vivo mutagenesis of the rock inhabitant Knufia petricola by a customized Ac/Ds transposon system N2 - 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. T2 - IUBMB Focused Meeting on Extremophilic Fungi (FUN-EX) CY - Ljubljana, Slovenia DA - 19.09.2023 KW - Black fungi KW - Genetics KW - Transposon KW - Mutagenesis PY - 2023 AN - OPUS4-58427 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Erdmann, Eileen T1 - Tools for Reverse and Forward Genetics in Knufia petricola N2 - Microcolonial black fungi are inhabitants of exposed natural and man-made surfaces in all climate zones. Genetic studies are hampered by slow growth, lacking sexual cycles and difficulties to transform these fungi. Recently, we implemented CRISPR/Cas9-based genetic tools enabling efficient targeted mutations of the rock inhabitant Knufia petricola (Eurotiomycetes/Chaetothyriales) for studying stress responses, the unusual types of cell division, mineral weathering and symbiotic interactions with phototrophic microbes (Voigt, Knabe et al. 2020, Sci Rep; Erdmann et al. 2022, Front Fungal Biol). To further explore the genome of this fungus as representative of the polyphyletic group of black fungi and to assign functions to yet unknown genes, a forward genetics approach is aimed. We chose the two-component Activator/ Dissociation (Ac/Ds) transposon system from maize for generating a collection of insertional mutants by in-vivo mutagenesis. For the optimal use of this genetic tool, an inducible promoter for the expression of the Ac transposase and by this the regulatable transposition of the resistance cassette-containing Ds transposon is requested. However, endogenous promoters for nitrate assimilation and galactose catabolism that are often used in fungi for regulatable gene expression are not inducible by their substrates in K. petricola suggesting that the regulatory networks for nutrient acquisition differ significantly in the oligotrophic black fungi. Therefore, the metabolism-independent Tet-on system was adopted and validated using a Ptet::gfp construct. No GFP fluorescence was detected in absence of the inducer doxycycline (DOX) while fluorescence intensities increased with increasing inducer concentrations. Consequently, the Tet-on system is suitable for regulatable expression of endogenous and foreign genes in K. petricola. T2 - VAAM Fachgruppentagung "Molecular Biology of Fungi" CY - Kaiserslautern, Germany DA - 07.09.2022 KW - Rock-inhabiting fungus KW - Genetic tools KW - Controllable gene expression PY - 2022 AN - OPUS4-55678 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Fuentes, Elsa A1 - Prieto, Beatriz T1 - Effect of climate change on phototrophic biofilms colonizing granitic rocks and their biodeteriogenic activity. N2 - Material cultural heritage from NW of the Iberian Peninsula is linked to the use or presence of granite, whether in historical buildings, sculptures, archaeological sites or any other artistic manifestation. This resistant material has the ideal physical characteristics that favor its longevity, but, like any material exposed to the environment, it is susceptible to deterioration. To this respect, environmental changes due to climate change and global change may modify biological colonization-substrate interactions. The main objective of the present research was to analyse the effects of Climate Change, more specifically changes in water availability, increased temperature, increased CO2 concentrations and changes in UV-B radiation patterns, on the survival and biodeteriogenic activity of phototrophic biofilms on granite heritage and the consequences for the built heritage. Both field and laboratory work were carried out. Field studies were conducted on three rural churches to characterise the current biological colonization. A higher proportion of algae than cyanobacteria was identified, with the genera Trentepohlia and Desmococcus standing out among the former. The most common current fungi are lichenised fungi, but black fungal genera such as Catenulostroma, Rhinocladiella and Knufia were also identified. Bacterial genera related to the production of sphinganes, EPS, carotenoids and some causing the so-called pink discolouration are also present. The effect of climate change on the growth and physiological state of the organisms was analysed in the laboratory. For this purpose, mixed biofilms were developed in the laboratory and exposed to changing conditions of temperature, water availability, CO2 and UV-B. A reduction in growth was observed with increasing water restriction, although in the initial colonization process, rock bioreceptivity was shown to be a more important aspect in favoring anchorage and retention of organisms. Temperature was shown to have a growth-enhancing effect when water availability was low, whereas an increase in CO2 only resulted in increased growth under conditions of high-water availability. Increased UV-B produced a reduction in growth, especially at the highest dose (equivalent to the current dose of a west-facing wall in southern Portugal, which is considered as high), while little difference in the physiological state of the organisms was observed at the current doses in Galicia (considered as medium) and southern Ireland (considered as low). Clear changes in microbial composition were observed, with a trend towards increased or greater resistance of cyanobacteria to higher temperature, lower water availability and higher UV-B doses. In addition, all these changes in development, physiological and microbial composition had an effect on the predominant pigments, tending towards more yellowish-brownish colourations, and on the ability of the organisms to generate biodeterioration by affecting ion concentrations on water solutions by enhancing their consumption or adhesion which lead to granite deterioration. Future work should take into account the combination of a greater number of climatic parameters simultaneously and also the possible resistances associated with the nature of the SAB community studied. T2 - 19th International Biodeterioration and Biodegradation Symposium CY - Berlin, Germany DA - 09.09.2024 KW - Biofilm KW - Granite KW - Climate change KW - Biodeterioration KW - Cultural heritage PY - 2024 AN - OPUS4-62271 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Gerrits, Ruben T1 - Fungal biofilms are supported by EPS and protective pigments in their substrate attachment and desiccation tolerance N2 - Rock-inhabiting fungi are known to colonise air-exposed substrates like minerals, photovoltaic panels building facades and monuments, withstanding the various stresses these extreme habitats are known for. Here we show how both colonisation and stress resistance are linked to the properties of the fungal cell surface. By deleting genes involved in the synthesis of melanin and carotenoid pigments of the model rock-inhabiting fungus Knufia petricola via CRISPR-Cas and comparing the behaviour of the gene-deletion mutants with the wild type (WT), we studied the role of these genes in mineral colonisation and stress sensitivity. The extracellular polymeric substances (EPS) of biofilms of the WT and mutants were extracted, quantified and chemically characterised. We observed that the absence of melanin affected the quantity and composition of the produced EPS: melanin-deficient mutants synthesised more EPS containing fewer pullulan-related glycosidic linkages. Moreover, in mineral dissolution experiments, these mutants showed a lower ability to colonise the mineral olivine. We hypothesise that not melanin, but the pullulan-related linkages enabled the melanin-producing strains to attach more strongly to the mineral. Moreover, ICP-OES analysis of the aqueous mineral-derived solutes showed that biofilms of the K. petricola mutants which could attach were able to dissolve the olivine faster than those that could not. The same mutants were also characterised by their sensitivity to desiccation stress: only the mutant deficient in both melanin and carotenoid synthesis was more sensitive to desiccation compared to the WT, indicating that a combination of both pigments is critical to withstand desiccation. Overall, these results show the critical role of the cell surface in the specific capacities of rock-inhabiting fungi. T2 - Biofilms 10 CY - Leipzig, Germany DA - 09.05.2022 KW - Bio-weathering KW - Desiccation KW - Fungal biofilms PY - 2022 AN - OPUS4-56675 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Gerrits, Ruben T1 - The role of melanin in fungal mineral weathering and metal corrosion N2 - 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. T2 - Goldschmidt 2023 Conference CY - Lyon, Frankreich DA - 10.07.2023 KW - MIC KW - Bio-weathering KW - Olivine PY - 2023 AN - OPUS4-58523 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Gerrits, Ruben T1 - Genetic engineering of melanised fungi to study the role of melanin in mineral weathering and metal corrosion N2 - Melanins are organic, dark pigments produced by many organisms. Fungi either fix these pigments in their cell wall or secrete them into their environment to protect themselves against an array of physicochemical stresses (e.g., UV irradiation, desiccation, ...). However, melanins can also adsorb metals like iron and therefore might have a role in the fungal capacity to deteriorate iron-containing minerals and metals. To study this, we focus on the fungi Knufia petricola, a rock-inhabiting fungus with melanised cell walls which deteriorates minerals, and Amorphotheca resinae, a soil-borne fungus able to secrete melanin, degrade alkane-containing fuels, and corrode metals. The broader significance of this work lies in the mitigation of climate change via the negative emission technology enhanced rock weathering for the former to the mitigation of corrosion of biofuel infrastructure for the latter. Genes of K. petricola involved in melanin synthesis and iron uptake were deleted and down- and up- regulated to generate strains with a different melanin content and ability to take up iron. These strains and the wild type (WT) were exposed to a growth solution containing the iron-magnesium silicate olivine of which dissolution rates were obtained by measuring aqueous Mg concentrations using ICP-OES. Thus, the specific mineral deterioration mechanisms of melanised fungi were identified. Abiotically, these experiments showed that iron oxidation at the surface inhibits olivine dissolution. K. petricola was able to enhance dissolution when this abiotic inhibition is strongest (at pH 6) but prevented dissolution when this inhibition is weakest (at pH 4). The fungus therefore dissolves olivine by interacting with the oxidised iron at the olivine surface. Its iron uptake pathways do not seem to be involved as mutants deficient in iron uptake dissolved olivine at the same rate as the WT. The higher dissolution rates of a mutant which secretes a melanin precursor and the lower rates of a melanin-deficient mutant, however, indicate that the iron-adsorbing and -reducing capacities of melanin play a key role. This hypothesis is further supported by carbon steel corrosion experiments with six strains of A. resinae isolated from anthropogenic and natural environments. Their varying ability to secrete melanin correlated with their corrosion rates. We now plan to develop CRISPR/Cas9-mediated genome editing techniques for A. resinae to figure out whether this correlation is also a causation. In summary, our data show that the iron-binding capacity of melanins enables fungi to deteriorate iron-containing substrates at a higher rate. T2 - IBBS19 CY - Berlin, Germany DA - 09.09.2024 KW - Black fungi KW - Weathering KW - Corrosion PY - 2024 AN - OPUS4-61171 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Gerrits, Ruben T1 - Quantifying and modelling growth of a subaerial black fungus N2 - Environmental, black fungi are known for their oligotrophic lifestyle and melanin production and are ubiquitous colonisers of natural and anthropogenic subaerial surfaces like marble monuments, washing machine soap dispensers and solar panels. Even though their growth is generally unappreciated, prevention or inhibition thereof is still to be accomplished. We have chosen the genetically amenable, black fungus Knufia petricola as a model species to study these organisms. Our goal is simple: the quantitative description of its growth on various environmental conditions to create a mathematical model using the iDynoMiCS 2.0 platform which could allow the development of a mitigation strategy. The conditions to be tested are the quantity and type of carbon and nitrogen sources, the pH and the presence of neighbouring colonies. First results showed that colonies grown on agar have the highest extension rates at pH 5, 0.01M NO3 or NH4, and 0.1M glucose or sucrose. Interestingly, extension rates but also CO2 production was unaffected by an increase in the concentration of the C-source from 0.01M to 0.1M. On a microscale, we observed that medium containing more C resulted in a higher quantity of branches produced by a single cell and not in a higher single cell growth rate. This rapidly results in a more compact microcolony versus the more extended colonies on C-deficient media. We believe that such deeper knowledge on the growth of black fungi will help mitigate their material colonisation and deterioration. T2 - IMC12 CY - Maastricht, Netherlands DA - 09.08.2024 KW - Black fungi KW - Modelling KW - Carbon and nitrogen limitation PY - 2024 AN - OPUS4-61170 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Gorbushina, Anna T1 - Life on the rocks and other hard surfaces N2 - 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 subaerial 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. We selected the rock-inhabiting fungus Knufia petricola (class Eurotiomycetes, order Chaetothyriales) that exhibits all the characteristics of microcolonial black fungi such as yeast-like cell growth, absence of reproductive structures and constitutive dihydroxynaphthalene (DHN) melanogenesis (Nai et al. 2013, Fungal Genet Biol). We developed protocols to efficiently generate and transform protoplasts resulting in stable homokaryotic transformants by targeting genes involved in pigment synthesis. The differences between the wild-type fungus and its melanin-deficient mutants were tested in geomicrobiological experiments and measured are now discussed in detail, with special accent on the possible effects of the mutation on EPS and other exuded substances. T2 - Departmental colloquium of the Tuscia University CY - Viterbo, Italy DA - 14.02.2020 KW - Ascomycetes KW - Fungal biofilms KW - Material surface colonisation KW - Genetics KW - Genomics KW - Extremophilic fungi PY - 2020 AN - OPUS4-50643 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia T1 - The roles of DHN melanin and the stress-activated MAP kinase in the rock inhabitant Knufia petricola N2 - Black fungi/yeasts exhibit high stress tolerance, yeast‑like or meristematic growth, and constitutive 1,8-dihydroxynaphthalene (DHN) melanin formation. Due to their slow growth, robust cell walls and the lack of sexual cycles and genetic tools, the underlying mechanisms of their phenotypic traits have remained largely unexplored. Using recently developed genetic tools, it is now possible to manipulate the genome of the rock-inhabiting model fungus Knufia petricola. Thus, gene functions and the cell biology of black fungi can be studied using CRISPR/Cas9-based genome editing and live-cell imaging with genetically encoded fluorescent proteins. Here, we are addressing the question to which extent constitutive pigment formation (melanin and carotenoids) and responses mediated by the stress-activated mitogen-activated protein (MAP) kinase contribute to the observed extremotolerance of K. petricola. The mutations of pks1, phs1 and both genes result in melanin-free (pink), carotenoid-free (black) and pigment-free (white) strains, respectively. The other putative melanogenic genes were identified in the genome, deleted to confirm their involvement in DHN melanogenesis and co-expressed in Saccharomyces cerevisiae for reconstruction of the synthesis pathway. Sak1 encoding the stress-activated MAP kinase was deleted in the wild-type and different pigment-deficient backgrounds. Growth of the obtained single, double and triple deletion mutants was tested by droplet tests on media supplemented with different stress-inducing agents. The Δsak1 mutants show slightly reduced growth rates even without environmental pressure and are hypersensitive to different stresses: e.g. osmotic, oxidative, membrane, pH and heat stress. Melanin-free Δsak1 mutants are more sensitive than black Δsak1 mutants to some but not all stress conditions, suggesting that melanin and the SAK1 pathway have complementary roles in protecting K. petricola from stress. T2 - 16th European Conference on Fungal Genetics CY - Innsbruck, Austria DA - 05.03.2023 KW - Black fungus KW - Extremotolerance KW - Pigments PY - 2023 AN - OPUS4-57147 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia T1 - Many-sided DHN melanin – spotlight on its function in microcolonial black fungi N2 - Dihydroxynaphthalene (DHN) 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. DHN melanogenesis is often regulated in a spatial and temporal fashion resulting e.g. in melanized reproduction, survival and/or infection structures. Remarkable is the regulation of the DHN melanogenesis in the foliar plant pathogen Botrytis cinerea: it involves two differently expressed PKSs providing the precursor in conidia and sclerotia, respectively (Schumacher 2016, Mol Microbiol). 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, DHN melanin builds a protective layer around all vegetative cells thus contributing to the survival of diverse environmental stresses even without specialized reproduction structures. As part of our continuing research on microcolonial rock-inhabiting fungi, we chose the genetically amenable Knufia petricola strain A95 (Nai et al. 2013, Fungal Genet Biol; Noack-Schönmann et al. 2014, AMB Express) for detailed studies. DHN-deficient mutants generated by targeted mutation of biosynthetic genes were studied with regard to the architecture of the cell wall and the EPS (extracellular polymeric substances) matrix, attachment to and weathering of olivine, as well as the tolerance to abiotic and biotic stresses. We will discuss the critical role of the outer cell surface (DHN melanin and EPS) in adhesion to the substrate and subsequent damage of the colonized surface. T2 - European Conference on Fungal Genetics (ECFG15) CY - Rome, Italy DA - 17.02.2020 KW - fungus KW - melanin KW - pigmentation PY - 2020 AN - OPUS4-50591 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia T1 - Versatile DHN melanin – spotlight on its function in microcolonial black fungi N2 - 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, a polyphyletic group of Ascomycetes (microcolonial fungi/ black yeasts) dwelling in hostile habitats such as bare rock surfaces in hot and cold deserts, exhibits 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 relevance of constitutive 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 DHN-deficient mutants are studied. Here, we will discuss the role of the DHN melanin layer on the outer cell wall in tolerating UV irradiation. T2 - Annual conference 2023 of the Association for General and Applied Microbiology CY - Göttingen, Germany DA - 10.09.2023 KW - Pigment KW - UV radiation KW - Tolerance PY - 2023 AN - OPUS4-58425 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia T1 - Rock-EATING FUNGI - Genetic Dive into the Biology of the Microcolonial Black Fungus Knufia petricola N2 - Microcolonial black fungi are a group of ascomycetes that exhibit high stress tolerance, yeast‑like growth and constitutive melanin formation. They dominate a range of hostile natural and man‑made environments, from desert rocks and salterns to dishwashers, roofs and solar panels. Due to their slow growth and a lack of genetic tools, the underlying mechanisms of black fungi’s phenotypic traits have remained largely unexplored. We chose to address this gap by genetically engineering the rock‑inhabiting fungus Knufia petricola (Eurotiomycetes, Chaetothyriales), a species that exhibits all characteristics of black fungi. The state‑of‑the‑art genetic toolkit, together with the annotated genome sequence of strain A95, firmly established K. petricola as a model for exploring microcolonial black fungi. T2 - VAAM-Jahrestagung 2021 CY - Online meeting DA - 18.03.2021 KW - Fungus KW - Genetics KW - Marble PY - 2021 AN - OPUS4-52496 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia T1 - Photoperception in plant- and rock-associated black fungi N2 - Fungi that share light-flooded habitats with phototrophs may profit from their excess photosynthetic products. Sunlight-associated stresses are however multiple: high temperatures, UV radiation with associated DNA damage, accumulation of reactive oxygen species (ROS), desiccation and osmotic stresses. Ascomycota dominating light-flooded habitats accurately sense and respond to changes in light using it as a cue to coordinate growth, stress responses as well as to establish pathogenic or symbiotic relationships. Two species from two light-flooded habitats – phyllosphere and sun-exposed solid surfaces – were analysed for their photoreceptor distribution. In both habitats phototroph-associated and black [dihydroxynaphthalene (DHN) melanin-containing] fungi are prevalent. This diversity was sampled with the plant-associated fungus Botrytis cinerea (Leotiomycetes), while Knufia petricola (Eurotiomycetes) was included as a typical biofilm-former on sun-exposed solid surfaces e.g. rocks, building facades, roofs, and solar panels. The analysis has shown that genomes of black fungi contain more photoreceptors than animal pathogens and saprophytes such as Aspergillus nidulans and Neurospora crassa1,2. B. cinerea that causes the grey mould disease by infecting the above-ground parts of more than 200 dicots has a highly sophisticated photosensory and signalling system that helps to avoid light and to locate susceptible hosts1. Rock-inhabiting Dothideomycetes and Eurotiomycetes including Knufia petricola possess equal numbers of photoreceptors along with the same set of protective metabolites i.e. melanin, carotenoids and mycosporines2. This similarity between black fungi from plant and rock surfaces suggests that photoperception and -regulation are important for sun-stressed fungi that receive nutrients through cooperation with phototrophs. CRISPR/Cas9-based genetic tools for manipulating K. petricola were established3 and are currently used for elucidating the functions of the different photoreceptors in the biology of rock-inhabiting fungi. This work was supported by the grant SCHU 2833/4-1 from the German Research Foundation (DFG) and internal funds of the BAM. T2 - 19th Congress of the European Society for Photobiology CY - Online meeting DA - 30.08.2021 KW - Fungi KW - Light KW - Extreme environments PY - 2021 AN - OPUS4-53191 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia T1 - Genetic manipulation of the microcolonial black fungus Knufia petricola N2 - Microcolonial black fungi, a polyphyletic group of ascomycetes, exhibit constitutive melanin formation, yeast-like growth and high stress tolerances. They dominate – often together with bacteria and algae in sub-aerial biofilms – a range of hostile environments including natural and man-made ones, from salterns to dishwashers, roofs and solar panels. Because of lacking genetic tools and the slow growth of most isolates, the genetic bases for these specific properties are largely unknown. The rock-inhabiting fungus Knufia petricola (Eurotiomycetes, Chaetothyriales) exhibits all characteristics of microcolonial black fungi and was selected as recipient for genetic engineering to study gene functions and genetic interactions. Different variants of green and red fluorescent proteins were successfully expressed indicating that fluorescence microscopy using genetically encoded fluorescent proteins and fluorescent dyes enables various cell biology approaches. Furthermore, genes of biosynthetic pathways (DHN melanin, carotenoids, uracil, adenine) were successfully mutated by applying traditional gene replacement and plasmid-based or ribonucleoprotein (RNP)-based CRISPR/Cas9 or silenced by RNA interference (RNAi). The availability of this advanced and efficient genetic toolbox and the annotated genome sequence of strain A95 makes K. petricola an excellent model for exploring the secrets of microcolonial black fungi. T2 - Departmental colloquium of the Tuscia University CY - Viterbo, Italy DA - 14.02.2020 KW - black fungus KW - genetics KW - Crispr/Cas9 PY - 2020 AN - OPUS4-50592 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Seeger, Stefan T1 - Analysis of air pollutants in ambient and indoor aerosolsby TXRF - application examples N2 - Quantitative chemical analysis of airborne particulate matter (PM) is vital for the understanding of health effects in indoor and outdoor environments, as well as for enforcing air quality regulations. Typically, airborne particles are sampled over long time periods on filters, followed by lab-based analysis, e.g., with inductively coupled plasma mass spectrometry (ICP-MS). Within the EURAMET EMPIR AEROMET project, cascade impactor aerosol sampling was combined for the first time with on-site total reflection X-ray fluorescence (TXRF) spectroscopy to develop a tool for quantifying particle element compositions within short time intervals and even on-site. This makes variations of aerosol chemistry observable with time resolution of only a few hours and with good size resolution in the PM10 range. A proof of principles of this methodological approach and the comparison to standard methods within the scope of a field campaign will be presented. Secondly, aerosol sampling and TXRF analysis seems suitable for the quantification of elements in indoor aerosols as well and may provide an important enhancement of existing methods for the analysis of organic species in aerosols (such as sampling and TD-GC/MS). As an example, the TXRF analysis of particles emitted from laser printers under controlled conditions in an environmental test chamber will be presented. T2 - TXRF Journal ClubB CY - Online meeeting DA - 24.02.2022 KW - Aerosol KW - TXRF KW - Cascade impactor KW - ICP-MS KW - Particles KW - Air quality monitoring KW - Element mass concentration PY - 2022 AN - OPUS4-54418 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - von Werder, Julia T1 - Biofilme auf Gebäuden - Biorezeptivität statt Biofilme N2 - Der Vortrag erläutert das Konzept der Gebäudebegrünung mit Biofilmen. Beton wird dabei als künstliches Gestein betrachtet, dessen Besiedelbarkeit durch Mikroorganismen gezielt verbessert werden soll. Ein dreistufiges Testsystem – Laborversuche, simulierte Bewitterung und Freilandtests – analysiert, wie physikalische und chemische Substrateigenschaften das Wachstum beeinflussen. Ein definierter Algen‑Pilz‑Modellbiofilm dient zur reproduzierbaren Bewertung der Besiedlung, erfasst über PAM‑Fluorometrie. Die Ergebnisse zeigen, dass Rauheit, Textur und Porosität entscheidend für Anhaftung, Feuchteretention und Biofilmwachstum sind. Zudem wird deutlich, dass die Organismen hohe Stresstoleranz benötigen, um variierende Umweltbedingungen an realen Fassaden zu überstehen. Insgesamt belegt das Projekt das Potenzial biorezeptiver Betone als low‑tech‑Fassadenbegrünung mit ökologischem Mehrwert. T2 - Workshop Ökologische Campusentwicklung: Wasser und Vegetationsmanagement in Helmholtz-Zentren CY - Potsdam, Germany DA - 03.04.2025 KW - Biofilm KW - Fassade KW - Biorezeptivität KW - Low-tech Begrünung KW - Modellbiofilm PY - 2025 AN - OPUS4-65448 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - von Werder, Julia T1 - Bioreceptive Building Facades: Codesigning with nature N2 - Building envelopes represent a key interface between solid substrates, the biosphere, and the atmosphere, and consequently serve as natural habitats for subaerial biofilms. This study investigates strategies to enhance the bioreceptivity of concrete cladding to support microalgae‑rich biofilms as a sustainable alternative to biocide‑dependent facade systems. Using a controlled dual‑species biofilm model and assessing algal vitality via PAM fluorometry, we examined how concrete composition and surface properties influence colonization dynamics. The results demonstrate that substrate pH and carbonation state are primary determinants of both biofilm establishment and organismal vitality, whereas nutrient supplementation exerts comparatively minor effects. Adhesion assays indicate that moderate mechanical stress can increase biofilm robustness, and laboratory rain simulations show that surface texture and near‑surface porosity promote water retention and enhance biofilm attachment. A synergistic interaction between the algal and fungal components was observed under specific stress conditions, offering a potential explanation for the reduced performance of biofilms when transitioning from ideal laboratory settings to outdoor environments. Overall, the findings highlight that the development of stable, photosynthetically active biofilm facades requires careful optimization of organism growth, physiological performance, and long‑term adhesion to the substrate. T2 - SASBE 2025: Smart and Sustainable Built Environment CY - Lille, France DA - 03.11.2025 KW - Concrete KW - Bioreceptivity KW - Laboratory test KW - Biofilm KW - Fluorometry PY - 2025 AN - OPUS4-65446 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - von Werder, Julia T1 - Greening Concrete with biofilms: Codesigning with nature N2 - uilding envelopes act as artificial lithic surfaces and natural substrates for subaerial biofilms, whose establishment depends on surface bioreceptivity. This study investigates how concrete cladding can be engineered to support microalgae‑dominated biofilms. A multi‑stage experimental framework—ranging from petri‑dish cultivation and adhesion testing to laboratory weathering simulations and outdoor exposure—was applied. A reproducible dual alga–fungus model biofilm enabled controlled assessment, with algal vitality quantified using PAM fluorometry. Results identify pH and carbonation as primary determinants of successful colonization, while nutrient addition has only minor influence. Adhesion tests show that moderate stress can enhance resilience to hydrodynamic forces. Rain‑simulation experiments highlight the importance of surface texture and near‑surface porosity for water retention, microbial attachment, and sustained growth. A synergistic interaction between algae and fungi under stress conditions may explain the poor outdoor performance of biofilms cultivated under ideal laboratory conditions. Overall, optimizing photosynthetic efficiency, organismal growth, and adhesion strength is essential for developing durable algal biofilm‑based façade systems. T2 - 4th International Conference of Sustainable Building Materials CY - Eindhoven, The Netherlands DA - 10.08.2025 KW - Concrete KW - Bioreceptivity KW - Laboratory test KW - Biofilm KW - Fluorometry PY - 2025 AN - OPUS4-65447 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - von Werder, Julia T1 - Gebäudebegrünung mit Biofilmen: Herausforderungen und Chancen N2 - Der Vortrag stellt die Ergebnisse der Forschungsarbeiten zur Biorezeptivität von Betonfassaden sowie der gezielten Applikation von Algen dominierten Biofilmen auf Betonfassaden vor. Insbesondere werden die Anforderungen an eine repräsentative Prüfmethode erläutert. T2 - BuGG-Tag der Forschung und Lehre Gebäudegrün 2024 CY - Leipzig, Germany DA - 26.09.2024 KW - Begrünung KW - Biorezeptivität KW - Beton KW - Biofilm PY - 2024 AN - OPUS4-62221 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - von Werder, Julia T1 - Living Concrete Walls: Engineering Bioreceptivity and Biofilms N2 - Building envelopes are a natural habitat of subaerial biofilms and can be more or less prone to be colonized (i.e. to be bioreceptive). Focusing on the added value of biofilms on manmade substrates represents new aesthetic frontiers and reduces the use of biocides. Moreover, the metabolic processes of photosynthetic biofilms can positively influence human health and life quality in densely populated cities by converting or absorbing pollutants. In the presented research the bioreceptivity of concrete claddings for building facades is engineered to sustain either natural or artificial establishment of microalgae-dominated biofilms. To be able to differentiate between the intrinsic material properties and the climatic boundary conditions, the experimental design in the first step comprised different analyses with model mono- and multi-species biofilms in sterile conditions and a high control of the environmental parameters. Growth and vitality of the algal component of the biofilms has been assessed with Pulse-amplitude modulation (PAM) fluorometry. T2 - 78th RILEM Annual Week & RILEM International Conference on Sustainable Materials & Structures: Meeting the major challenges of the 21st century - SMS 2024 CY - Toulouse, France DA - 25.08.2024 KW - Bioreceptivity KW - Biofilm KW - Weathering KW - Concrete KW - PAM fluorometry KW - Carbonation PY - 2024 AN - OPUS4-62224 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -