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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.
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. A cell biological approach was taken by generating K. petricola strains expressing green or red fluorescent protein variants. By applying: (1) traditional gene replacement; (2) gene editing and replacement via plasmid-based or ribonucleoprotein (RNP)-based CRISPR/Cas9, and (3) silencing by RNA interference (RNAi), we constructed mutants in the pathways leading to melanin, carotenoids, uracil and adenine. Stable single and double mutants were generated with homologous recombination (HR) rates up to 100%. Efficient, partially cloning-free strategies to mutate multiple
genes with or without resistance cassettes were developed. This 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.
Genetics for geomycology
(2017)
Black microcolonial fungi (MCF) are persistent inhabitants of rock surfaces. Biofilm establishment and more importantly, quantification of its geological input are so far only possible to be studied in simplified, well-controlled laboratory experiments. Therefore, the ability of MCF to cope with multiple, rapidly fluctuating stresses makes the group an interesting subject in the study of stress resistance but also in mineral weathering processes. We studied the role of protective pigments in stress resistance and mineral weathering of the rock-inhabiting fungus Knufia petricola (Chaetothyriales) strain A95.
Fungal melanins are distinctive markers of rock-inhabiting ascomycetes. These complex polyphenols play important roles in stress tolerance while being essential components of fungal cell walls and useful biomarkers. Here we report signatures of melanins and carotenoids in pigment mutant strains of the black yeast Knufia petricola A95 using Raman spectroscopy
Mediaeval stained glass has been treated with Polymethylmetacrylate coatings by Kwiatkowski in Poland during the 1950th. Such treated panels were found in the Johannis Church of Toruń (without protective glazing), in the Cathedral of Włocławek (behind a protective glazing), and on glass kept in exhibition cases in the museum of Toruń. Surface coatings have been detected and analyzed. There was no extensive contamination by fungi or bacteria if the glass was either coated or not.
Black ascomycetous microcolonial fungi (MCF) are persistent inhabitants of rock surfaces in deserts as well as ubiquitous in other wide-spread terrestrial ecosystems including man-made materials such as solar panels. The ability of MCF to cope with multiple, rapidly fluctuating stresses makes the group an interesting subject in the study of stress resistance. Carotenoids and melanin are amongst the protective pigments that contribute to the robustness of MCF. We studied their role in stress resistance of the non-pathogenic rock-inhabiting fungus Knufia petricola (Chaetothyriales) strain A95. Disruption of melanin synthesis affects not only oxidative-stress resistance but also the extracellular polysaccharide (EPS) matrix and lipid composition of the cell membrane.
Black ascomycetous microcolonial fungi (MCF) are persistent inhabitants of rock surfaces in deserts as well as ubiquitous in other wide-spread terrestrial ecosystems including man-made materials such as solar panels. The ability of MCF to cope with multiple, rapidly fluctuating stresses makes the group an interesting subject in the study of stress resistance. Carotenoids and melanin are amongst the protective pigments that contribute to the robustness of MCF. We studied their role in stress resistance of the non-pathogenic rock-inhabiting fungus Knufia petricola (Chaetothyriales) strain A95. Disruption of melanin synthesis affects not only oxidative-stress resistance but also the extracellular polysaccharide (EPS) matrix and lipid composition of the cell membrane.
Territories of rock-inhabiting fungi: Survival on and alteration of solid air-exposed surfaces
(2018)
Subaerial biofilms that are omnipresent at the interface between all solid substrates and the atmosphere are composed of a unique and widespread group of ascomycetes called rock-inhabiting fungi or microcolonial fungi (MCF), typically in communities with other microorganisms. While subaerial biofilms in toto have important roles in mineral weathering and biodeterioration of materials, methodological approaches to subaerial biofilm communities are diverse and frequently focussed on MCF. Here, we review the historical development of the research methods applied in the field and consider perspectives to increase our understanding of the biofilm-induced changes of solid substrate surfaces.
Soil formation on rock surfaces is intrinsically coupled to primary microbial colonisation of the atmosphere-lithosphere interface. Rock-inhabiting microbial life is ubiquitous but the mechanisms of biofilm establishment and more importantly, quantification of its geological input are so far only possible to be studied in simplified, well-controlled laboratory experiments. In a previous study [1] a laboratory biofilm consisting of the phototrophic cyanobacterium Nostoc punctiforme ATCC 29133 and the rock-inhabiting ascomycete Knufia petricola A95 was tested for its mineral weathering potential. Mineral dissolution was enhanced in biotic experiments as compared to abiotic ones. Here the influence of K. petricola strain A95 and a recently constructed melanin deficient mutant (A95ΔPKS) were used to study the influence of fungal pigments on weathering of forsteritic olivine. The olivine with fungal biomass was submerged in a nutrient solution (pH 6.2) in batch-reactor flasks that were incubated for 90 d at 25°C and 90 μmol photons.m2.sec-1, while shaken at 150 rpm. qPCR was used to quantify the growth of fungi. Mineral dissolution was quantified by ICP-OES analysis of the liquid medium while SEM-EDX analysis of the solid phase was used to determine secondary mineral formation and visualise growth behaviour. Wild type and mutant accelerated dissolution of the mineral: over time both release more Mg and Si from olivine than the abiotic control. Also SEM revealed a closer physical contact of the wild type cells to the mineral and a higher production of EPS of the melanin mutant A95ΔPKS. This important difference in the ability of the wild type strain to adhere to the mineral surface might be crucial in maintaining a biologically modified environment. This biologically engineered habitat serves as a place where mineral dissolution as well as deposition of metabolic products (EPS + pigments) can impact the rock surface. We expect this study to increase the awareness on the impact of microbiology, and more specifically, rock-inhabiting fungi on mineral weathering. [1] Seiffert, F., Bouchez, J., von Blanckenburg, F., and Gorbushina, A. A. (2014). Microbial colonization of bare rocks: laboratory biofilm enhances mineral weathering. Proc. Earth Plan. Sci. 12,123–129.doi: 10.1016/j.proeps.2014.08.042