TY - CONF A1 - Gerrits, Ruben A1 - Schumacher, Julia A1 - Gorbushina, Anna 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 - CONF A1 - Gerrits, Ruben A1 - Hennehan, M.J. A1 - Frick, D.A. A1 - von Blanckenburg, F. A1 - Schumacher, Julia A1 - Gorbushina, Anna 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 - 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 - JOUR A1 - Gerrits, Ruben A1 - Wirth, R. A1 - Schreiber, A. A1 - Feldmann, Ines A1 - Knabe, Nicole A1 - Schott, J, A1 - Benning, L.G. A1 - Gorbushina, Anna T1 - High-resolution imaging of fungal biofilm-induced olivine weathering JF - Chemical Geology N2 - Many microorganisms including free-living and symbiotic fungi weather minerals through the formation of biofilms on their surface. Weathering thus proceeds not only according to the mineral’s chemistry and the environmental conditions but also according to the local biofilm chemistry. These processes can be dissected in experiments with defined environmental settings and by employing genetic tools to modify traits of the fungal biofilm. Biofilms of the rock-inhabiting fungus Knufia petricola strain A95 (wild-type, WT) and its melanin-deficient mutant (ΔKppks) were grown on polished olivine sections in subaerial (air-exposed) and subaquatic (submerged) conditions. After seven months of interaction at pH 6 and 25°C, the fungus-mineral interface and abiotic olivine surface were compared using high resolution transmission electron microscopy (HRTEM). The abiotic, subaquatic olivine section showed a 25 nm thick, continuous amorphous layer, enriched in Fe and depleted in Si compared to the underlying crystalline olivine. This amorphous layer formed either through a coupled interfacial dissolution reprecipitation mechanism or through the adsorption of silicic acid on precipitated ferric hydroxides. Its thickness was likely enhanced by mechanical stresses of polishing. Directly underneath a fungal biofilm (WT and mutant alike), the surface remained mostly crystalline and was strongly etched and weathered, indicating enhanced olivine dissolution. The correlation between enhanced olivine dissolution and the absence of a continuous amorphous layer is a strong indication of the dissolution-inhibiting qualities of the latter. We propose that the fungal biofilm sequesters significant amounts of Fe, preventing formation of the amorphous layer and driving olivine dissolution onwards. The seemingly similar olivine surface underneath both WT and mutant biofilms illustrates the comparably insignificant role of specific biofilm traits in the weathering of olivine once biofilm attachment is imposed. Under subaerial conditions, the absence of water on the abiotic surface prohibited olivine dissolution. This was overcome by the water retention capacities of both the WT and mutant biofilm: the olivine surface underneath subaerial fungal biofilms was as weathered as the corresponding subaquatic olivine surface. Under the studied environmental settings, the effect of fungal biofilms on olivine weathering seems to be universal, independent of the production of melanin, the composition of extracellular polymeric substances (EPS) or air-exposure. KW - Bio-weathering KW - Forsterite KW - Extracellular polymeric substances KW - Melanin KW - Black fungi PY - 2021 DO - https://doi.org/10.1016/j.chemgeo.2020.119902 VL - 559 SP - 119902 PB - Elsevier B.V. AN - OPUS4-51403 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CHAP A1 - Lessovaia, S.N. A1 - Gerrits, Ruben A1 - Gorbushina, Anna A1 - Polekhovsky, Y.S. A1 - Dultz, S. A1 - Kopitsa, G.G. ED - Frank-Kamenetskaya, O.V. ED - Vlasov, D. ED - Panova, E.G. ED - Lessovaia, S.N. T1 - Modeling Biogenic Weathering of Rocks from Soils of Cold Environments T2 - Processes and Phenomena on the Boundary Between Biogenic and Abiogenic Nature N2 - Morphologically simple and microbially dominated ecosystems termed “biofilms” have existed on Earth for a long period of biosphere evolution. A model biofilm combining one heterotroph and one phototroph component was used in a laboratory experiment to simulate biogenic weathering with two different specimens of basic rock samples from the soil profiles. The rocks fragments from the regions of cold environments of Eurasia,where abiotic physical processes, including rock disintegration initiated by freezing–thawing cycles, represent the most probable Scenario of rock weathering, were subjected to biological colonization. The rock Fragments were represented by dolerite and metagabbro amphibolites. Polished sections of the rock samples were inoculated with the model microbiological consortium of the oligotrophic fungus and the phototrophic cyanobacteria (biofilm). After 3 month runtime of the experiment the progress of rock weathering was derived from the growth of the biofilm on the rock surfaces. The model biofilm visualization on the rock surface of polished sections illustrated their stronger development namely on dolerite in comparison with metagabbro amphibolite. The findings confirmed the higher sensitivity of dolerite to biogenic weathering due to (i) mineral association, in which quartz was absent and (ii) porosity providing higher specific surface area for biotic—abiotic interaction influenced by the occurrence of micro-porosity in the rock. KW - Biogenic weathering KW - Rock leaching KW - Fractal structure KW - Biofilm formation KW - Internal pores PY - 2020 DO - https://doi.org/10.1007/978-3-030-21614-6_27 SP - 501 EP - 515 PB - Springer AN - OPUS4-51442 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Pokharel, R. A1 - Gerrits, Ruben A1 - Schuessler, J. A1 - von Blanckenburg, F. T1 - Mechanisms of olivine dissolution by rock-inhabiting fungi explored using magnesium stable isotopes JF - Chemical Geology N2 - To unravel the dissolution mechanisms of olivine by a rock-inhabiting fungus we determined the stable isotope ratios of Mg on solutions released in a laboratory experiment. We found that in the presence of the fungus Knufia petricola the olivine dissolution rates were about seven-fold higher (1.04×10−15 mol cm−2 s−1) than those in the abiotic experiments (1.43×10−16 mol cm−2 s−1) conducted under the same experimental condition (pH 6, 25 °C, 94 days). Measured element concentrations and Mg isotope ratios in the supernatant solutions in both the biotic and the abiotic experiment followed a dissolution trend in the initial phase of the experiment, characterized by non-stoichiometric release of Mg and Si and preferential release of 24Mg over 26Mg. In a later phase, the data indicates stoichiometric release of Mg and Si, as well as isotopically congruent Mg release. We Attribute the initial non-stoichiometric phase to the rapid replacement of Mg2+ in the olivine with H+ along with simultaneous polymerization of Si tetrahedra, resulting in high dissolution rates, and the stoichiometric phase to be influenced by the accumulation of a Si-rich amorphous layer that slowed olivine dissolution. We attribute the accelerated dissolution of olivine during the biotic experiment to physical attachment of K. petricola to the Si-richamorphous layer of olivine which potentially results in ist direct exposure to protons released by the fungal cells. These additional protons can diffuse through the Si-rich amorphous layer into the crystalline olivine. Our results also indicate the ability of K. petricola to dissolve Fe precipitates in the Si-rich amorphous layer either by protonation, or by Fe(III) chelation with siderophores. Such dissolution of Fe precipitates increases the porosity of the Si-rich amorphous layer and hence enhances olivine dissolution. The acceleration of mineral dissolution in the presence of a rock-dissolving fungus further suggests that its presence in surficial CO2 sequestration plants may aid to accelerate CO2 binding. KW - Olivine KW - Magnesium KW - Isotopes KW - Fungus PY - 2019 DO - https://doi.org/10.1016/j.chemgeo.2019.07.001 SN - 0009-2541 SN - 1872-6836 VL - 525 SP - 18 EP - 27 PB - Elsevier AN - OPUS4-48824 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Gerrits, Ruben A1 - Pokharel, R. A1 - Breitenbach, Romy A1 - Radnik, Jörg A1 - Wirth, R. A1 - Schuessler, J. A. A1 - Benning, L. G. A1 - von Blanckenburg, F. A1 - Gorbushina, Anna A1 - Feldmann, Ines 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 - JOUR A1 - Pokharel, R. A1 - Gerrits, Ruben A1 - Schuessler, J. A. A1 - Frings, P. J. A1 - Sobotka, R. A1 - Gorbushina, Anna A1 - von Blanckenburg, F. T1 - Magnesium stable isotope fractionation on a cellular level explored by cyanobacteria and black fungi with implications for higher plants JF - Environmental Science & Technology N2 - In a controlled growth experiment we found that the cyanobacterium Nostoc punctiforme has a bulk cell 26Mg/24Mg ratio (expressed as δ26Mg) that is −0.27‰ lower than the growth solution at a pH of ca. 5.9. This contrasts with a recently published δ26Mg value that was 0.65‰ higher than growth solution for the black fungus Knufia petricola at similar laboratory conditions, interpreted to reflect loss of 24Mg during cell growth. By a mass balance model constrained by δ26Mg in chlorophyll extract we inferred the δ26 Mg value of the main Mg compartments in a cyanobacteria cell: free cytosolic Mg (−2.64‰), chlorophyll (1.85‰), and the nonchlorophyll-bonded Mg compartments like ATP and ribosomes (−0.64‰). The lower δ26Mg found in Nostoc punctiforme would thus result from the absence of significant Mg efflux during cell growth in combination with either (a) discrimination against 26Mg during uptake by desolvation of Mg or transport across protein channels or (b) discrimination against 24Mg in the membrane transporter during efflux. The model predicts the preferential incorporation of 26Mg in cells and plant organs low in Mg and the absence of isotope fractionation in those high in Mg, corroborated by a compilation of Mg isotope ratios from fungi, bacteria, and higher plants. KW - Cyanobacteria KW - Black fungi KW - Nostoc punctiforme KW - Knufia petricola KW - Magnesium PY - 2018 DO - https://doi.org/10.1021/acs.est.8b02238 SN - 1520-5851 SN - 0013-936X VL - 52 IS - 21 SP - 12216 EP - 12224 PB - ACS Publications AN - OPUS4-46832 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Rasesh, Pokharel A1 - Gerrits, Ruben A1 - Schuessler, Jan A. A1 - Floor, Geerke H. A1 - Gorbushina, Anna A1 - von Blanckenburg, Friedhelm T1 - Mg isotope fractionation during uptake by a rock-inhabiting, model microcolonial fungus Knufia petricola at acidic and neutral pH JF - Environmental Science and Technology N2 - The model rock-inhabiting microcolonial fungus Knufia petricola fractionates stable Mg isotopes in a time and pH-dependent manner. During growth, the increase of 26Mg/24Mg in the fungal cells relative to the growth media amounted to 0.65 ± 0.14‰at pH 6 and 1.11 ± 0.35‰at pH 3. We suggest a constant equilibrium fractionation factor during incorporation of Mg into ribosomes and ATP as a cause of enrichment of 26Mg in the cells. We suggest too that the proton gradient across the cell wall and cytoplasmic Membrane controls Mg2+ transport into the fungal cell. As the strength of this gradient is a function of extracellular solution pH, the pHdependence on Mg isotope fractionation is thus due to differences in fungal cell mass fluxes. Through a mass balance model we show that Mg uptake into the fungal cell is not associated with a unique Mg isotope fractionation factor. This Mg isotope fractionation dependence on pH might also be observed in any organism with cells that follow similar Mg uptake and metabolic pathways and serves to reveal Mg cycling in ecosystems. KW - Isotope fractionation KW - Fungus KW - Magnesium PY - 2017 DO - https://doi.org/10.1021/acs.est.7b01798 SN - 0013-936X SN - 1520-5851 VL - 51 IS - 17 SP - 9691 EP - 9699 PB - ACS Publications CY - USA AN - OPUS4-43018 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Gerrits, Ruben A1 - Pokharel, R. A1 - Feldmann, Ines A1 - Schüssler, J.A. A1 - Toepel, Jörg A1 - von Blanckenburg, F. A1 - Gorbushina, Anna T1 - Olivine dissolution by a model consortium: biological impact and analytical methodology considerations N2 - Microbiological biofilms on rocks are ubiquitous in nature and their influence on soil formation through rock weathering has been shown (Gorbushina 2007). However, most previous studies on rock weathering are limited to understanding the physical and chemical aspects overlooking the impact of biota. Due to the enormous amounts of variables that come with a biological process, the quantification of its influence is only possible by using well-controlled and simplified laboratory models. Thereby gaining more insight on the impact of rock inhabiting biofilms on mineral weathering. This presentation will show the impact of biotic weathering in terms of olivine dissolution rates Natural forsterite was incubated in batch reactor flasks with and without a model consortium consisting of the phototrophic cyanobacterium Nostoc punctiforme and the rock-inhabiting ascomycete Knufia petricola, and submerged in a growth solution (pH 6). The flasks were incubated for 30 days under 25°C, 90 µmol photons/m2s and were shaken at 150 rpm. qPCR was performed to quantify the cell number of both organisms, BET to gather the specific surface of the used olivine and ICP-OES to follow up the change of concentration of the leached out metals. Our results show that our model consortium, especially K. petricola does increase the dissolution rate of olivine. The pH increased from the initial 6 to around 7.2 for all setups. Initially Mg was preferentially released over Si (Mg/Si of 3.5), until after two days the ratio starts equilibrating around stoichiometric dissolution. During this timeframe the dissolution rate drops by nearly two orders of magnitude, just as observed by Daval et al., (2011). The difference in dissolution rates between the different setups is initially non-existent, but increases over time. After 30 days the setup with K. petricola gives a dissolution rate of 1.08 10-13 moles/cm2s, compared to 9.23 10-14 moles/cm2s for the abiotic setup. We expect this study to cause awareness on the impact of microbiology on mineral weathering. Additionally it is a starting point for other, more complicated experiments using for instance flow through or drip flow reactors or other minerals. T2 - VAAM CY - Jena, Germany DA - 13.03.2016 KW - Olivine KW - Weathering KW - Biofilm PY - 2016 AN - OPUS4-37971 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -