TY - CONF A1 - Gerrits, Ruben T1 - Genetic engineering of a black rock-inhabiting fungus shows its strengths and weaknesses  N2 - Environmental black fungi are known to inhabit anthropogenic habitats like marble monuments, solar panels, indoor walls and even washing machines. These pests are generally assumed to persist and grow in these environments due to their melanin production and oligotrophic lifestyle, respectively. We aimed to confirm these assumptions and to find methods to mitigate their substrate colonisation and deterioration by looking at their ability to produce melanin and take up iron, the latter being a general weakness of aerobes. This was done by means of selecting one species, Knufia petricola, and applying CRISPR/Cas9-mediated genome editing techniques. This showed that K. petricola produces one siderophore; deletion of its encoding gene showing no strong phenotypic effects. Deletion of the other iron uptake mechanism – reductive iron assimilation (RIA) – did however cause a strong growth deficiency at iron depleted conditions. Both together reveal that K. petricola has a weak siderophore and relies on RIA to acquire iron, reflecting a natural habitat free of competition for iron. This comes at a price: K. petricola but also other black fungi isolated from solar panels are extremely sensitive to mediocre iron chelators, offering us a mitigation strategy. Melanin, although adsorbing and reducing iron, did not have an observable effect on iron uptake. The pigment did however affect the fungal ability to withstand desiccation: pigment-deficient mutants were not able to withstand a prolonged incubation in a desiccator. This is a clear advantage in the colonisation of dry subaerial surfaces. T2 - 12th International Mycological Congress (IMC12) CY - Maastricht, Netherlands DA - 12.08.2024 KW - Iron uptake KW - Black fungi KW - Melanin PY - 2024 AN - OPUS4-62269 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Gerrits, Ruben T1 - Olivine weathering by the genetically amenable rock-inhabiting fungus Knufia petricola A95 N2 - In arid sun-exposed environments, rock-inhabiting fungi dominate microbial communities on rock surfaces. Fungi are known to enhance rock weathering, although the exact mechanisms are not completely understood. These natural processes can be simulated by reproducible experimental systems. Particularly attractive are genetically amenable rock-weathering fungi, where certain traits can be knocked-out to identify weathering-relevant functions. Here, we studied the effect of the rock-inhabiting fungus Knufia petricola A95 and its melanin-deficient mutant (ΔKppks) on the weathering of a Fe-bearing olivine. We examined the olivine dissolution kinetics and looked at the olivine-fungus interface. For the former we inoculated olivine powder with the fungus in batch and mixed flow reactors and analysed the medium solution by ICP-OES, for the latter biofilm-covered olivine sections from flow-through reactors were analysed by FIB-TEM. In general olivine dissolution was lower when olivine was incubated without fungi: the abiotic olivine dissolution rates were lowest and the surface of the abiotically reacted olivine sections showed no etching. Various chemical analyses show Fe (oxyhydr)oxide precipitation on top of the abiotically reacted olivine, indicating its role in inhibiting olivine dissolution. Both the wild type (WT) and ΔKppks solubilised and bound significant amounts of Fe released by olivine dissolution. Fe (oxyhydr)oxide precipitation was thus reduced, explaining the enhanced olivine dissolution in their presence. Particularly efficient at preventing Fe precipitation were attached WT cells: the inhibition of olivine dissolution almost completely disappeared. The attachment capacity of the WT is most likely caused by the production of WT-specific extracellular polymeric substances. Our presented experimental systems allowed the precipitation of Fe (oxyhydr)oxides and included a rock-inhabiting fungus and thus simulated weathering processes relevant to natural ecosystems. T2 - ISEB 2019 CY - Potsdam, Germany DA - 23.09.2019 KW - Olivine KW - Weathering KW - Fungus PY - 2019 AN - OPUS4-49585 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 - Gerrits, Ruben T1 - Iron uptake by the rock-inhabiting fungus Knufia petricola N2 - Iron is arguably the most essential metal in living organisms. For rock-inhabiting fungi its acquisition might be unconventional as they (1) tend to inhabit iron-deficient, oxygen-rich surfaces like marble monuments and solar panels and (2) produce the black, iron-adsorbing pigment melanin. We used a range of analytical methods, ongoing mineral dissolution experiments and gene deletion mutants of the model rock-inhabiting fungus Knufia petricola to figure out the mechanisms and substrate deteriorating effects of iron uptake by these organisms. To study both siderophore-mediated and reductive iron assimilation (RIA), genes like sidC, encoding a putative siderophore synthetase and ftr1 and fet3 encoding the subunits of an iron permease-oxidase were deleted. At iron deficient conditions, growth of the wild type (WT) and ΔsidC mutant was similar, whereas growth of the Δftr1-fet3 mutant and the double mutant ΔsidC/Δftr1-fet3 was diminished and absent, respectively. We were not able to detect the siderophore of K. petricola and the WT and mutants were not able to grow at low concentrations of strong iron chelators. However, in a cross-feeding experiment, an overexpression strain of sidC allowed more growth of ΔsidC/Δftr1-fet3 on iron deficient medium than the WT, whereas the ΔsidC mutant could not do so at all. Compared to the WT, the sidC overexpression strain also withstood oxidative stress better and had a shorter lag time and higher growth rate. Combined, these results indicate that K. petricola relies more on RIA than siderophore-mediated uptake as it likely excretes low quantities of a primarily intracellular siderophore. Interestingly, Δftr1-fet3 had a higher iron content than the WT at iron deficient conditions. This difference disappeared upon deletion of melanin synthesis (Δpks1 vs. Δpks1/Δftr1-fet3): melanin-bound iron can likely not be used without RIA. K. petricola’s chelation incapacity implies a habitat free of competition for iron while offering us a mitigation strategy. T2 - ECFG16 CY - Innsbruck, Austria DA - 05.03.2023 KW - Siderophore KW - Melanin KW - Reductive iron assimilation PY - 2023 AN - OPUS4-57148 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Gerrits, Ruben T1 - Data of "An experimental study of fungal olivine weathering" N2 - This file contains all the data used for the figures shown in the Dissertation of Ruben Gerrits with the title "An experimental study of fungal olivine weathering". In this study, the weathering-affecting, rock-inhabiting fungus, Knufia petricola A95 and the Fe-bearing olivine (Fe0.2Mg1.8SiO4) were selected to investigate fungi-induced effects on mineral dissolution. The availability of a melanin-deficient mutant (ΔKppks) of K. petricola A95, that produced more extracellular polymeric substances (EPS) than the wild type (WT), enabled comparative studies of the role of melanin and EPS in weathering processes. KW - Olivine KW - Weathering KW - Fungus PY - 2019 DO - https://doi.org/10.26272/opus4-48770 PB - Bundesanstalt für Materialforschung und -prüfung (BAM) CY - Berlin AN - OPUS4-48770 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Catanzaro, Ilaria A1 - Gerrits, Ruben A1 - Feldmann, Ines A1 - Gorbushina, Anna A1 - Onofri, Silvano A1 - Schumacher, Julia T1 - Deletion of the polyketide synthase‐encoding gene pks1 prevents melanization in the extremophilic fungus Cryomyces antarcticus N2 - Cryomyces antarcticus, a melanized cryptoendolithic fungus endemic to Antarctica, can tolerate environmental conditions as severe as those in space. Particularly, its ability to withstand ionizing radiation has been attributed to the presence of thick and highly melanized cell walls, which—according to a previous investigation—may contain both 1,8‐dihydroxynaphthalene (DHN) and L‐3,4 dihydroxyphenylalanine (L‐DOPA) melanin. The genes putatively involved in the synthesis of DHN melanin were identified in the genome of C. antarcticus. Most important is capks1 encoding a non‐reducing polyketide synthase (PKS) and being the ortholog of the functionally characterized kppks1 from the rock‐inhabiting fungus Knufia petricola. The co‐expression of CaPKS1 or KpPKS1 with a 4′‐phosphopantetheinyl transferase in Saccharomyces cerevisiae resulted in the formation of a yellowish pigment, suggesting that CaPKS1 is the enzyme providing the precursor for DHN melanin. To dissect the composition and function of the melanin layer in the outer cell wall of C. antarcticus, non‐melanized mutants were generated by CRISPR/Cas9‐mediated genome editing. Notwithstanding its slow growth (up to months), three independent non‐melanized Δcapks1 mutants were obtained. The mutants exhibited growth similar to the wild type and a light pinkish pigmentation, which is presumably due to carotenoids. Interestingly, visible light had an adverse effect on growth of both melanized wild‐type and non‐melanized Δcapks1 strains. Further evidence that light can pass the melanized cell walls derives from a mutant expressing a H2B‐GFP fusion protein, which can be detected by fluorescence microscopy. In conclusion, the study reports on the first genetic manipulation of C. antarcticus, resulting in non‐melanized mutants and demonstrating that the melanin is rather of the DHN type. These mutants will allow to elucidate the relevance of melanization for surviving extreme conditions found in the natural habitat as well as in space. KW - Astrobiology KW - Black fungi KW - CRISPR/Cas9 KW - DHN melanin KW - Cryptoendolithism PY - 2024 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-606599 DO - https://doi.org/10.1002/iub.2895 SN - 1521-6551 VL - 76 IS - 12 SP - 1072 EP - 1090 PB - Wiley AN - OPUS4-60659 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Breitenbach, Romy A1 - Gerrits, Ruben A1 - Dementyeva, Polina A1 - Knabe, Nicole A1 - Schumacher, Julia A1 - Feldmann, Ines A1 - Radnik, Jörg A1 - Ryo, M. A1 - Gorbushina, Anna T1 - The role of extracellular polymeric substances of fungal biofilms in mineral attachment and weathering N2 - The roles extracellular polymeric substances (EPS) play in mineral attachment and weathering were studied using genetically modified biofilms of the rock-inhabiting fungus Knufia petricola strain A95. Mutants deficient in melanin and/or carotenoid synthesis were grown as air-exposed biofilms. Extracted EPS were quantified and characterised using a combination of analytical techniques. The absence of melanin affected the quantity and composition of the produced EPS: mutants no longer able to form melanin synthesised more EPS containing fewer pullulan-related glycosidic linkages. Moreover, the melanin-producing strains attached more strongly to the mineral olivine and dissolved it at a higher rate. We hypothesise that the pullulan-related linkages, with their known adhesion functionality, enable fungal attachment and weathering. The released phenolic intermediates of melanin synthesis in the Δsdh1 mutant might play a role similar to Fe-chelating siderophores, driving olivine dissolution even further. These data demonstrate the need for careful compositional and quantitative analyses of biofilm-created microenvironments. KW - Biofilms PY - 2022 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-549025 DO - https://doi.org/10.1038/s41529-022-00253-1 SN - 2397-2106 VL - 6 SP - 1 EP - 11 PB - Springer Nature CY - London AN - OPUS4-54902 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Breitenbach, Romy A1 - Gerrits, Ruben A1 - Dementyeva, Polina A1 - Knabe, Nicole A1 - Schumacher, Julia A1 - Feldmann, Ines A1 - Radnik, Jörg A1 - Ryo, M. A1 - Gorbushina, Anna T1 - Data for "The role of extracellular polymeric substances of fungal biofilms in mineral attachment and weathering" N2 - Data for the publication "The role of extracellular polymeric substances of fungal biofilms in mineral attachment and weathering" (https://doi.org/10.1038/s41529-022-00253-1). It includes: - The Summary of the EPS concentration, EPS sugar components and EPS linkages. - The Summary of the XPS analysis of freeze-dried biofilm samples of all strains. - The Summary of the pH, Mg, SI and Fe concentration, biomass and olivine dissolution rate for each time point of all dissolution experiments. KW - Biofilms PY - 2022 DO - https://doi.org/10.26272/opus4-54901 PB - Bundesanstalt für Materialforschung und -prüfung (BAM) CY - Berlin AN - OPUS4-54901 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -