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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.
Simple, microbial biofilms have prevailed since life began on Earth. Biofilms form at the interfaces of solids with gases or liquids and have multiple effects on Substrate and element cycles. In geobiological terms, the most interesting microbial communities are those that form on solids exposed to air (subaerial). Microbial colonisers of the atmosphere-lithosphere interface include algae, cyanobacteria, fungi as well as heterotrophic bacteria and they have colonised virtually every rock surface throughout the entire geological history of the Earth. In addition to sequestering carbon, sub-aerial biofilms (SABS) actively participate in rock weathering. Rock-inhabiting SABs are the primary settlers on lava following volcanic eruptions and on rocks following the retreat of glaciers. SABs especially dominate hostile environments in which growth of higher Vegetation is restricted especially in deserts, polar- and alpine regions. SABs are the primary colonisers of lithospheric (e.g. rocks) and anthropogenic Substrates (buildings, monuments, solar panels, etc.). Life at the solid material/atmosphere interface influences and is affected by both the underlying Substrate and the microclimate surrounding it. Although subaerial life is ubiquitous, how SABs develop and importantly degrade underlying Substrates can only be clarified in well-controlled experiments that often involve simplified model Systems, So far, biofilm development on solar panels has been studied using; (i) metagenomics; (ii) in situ microscopy; and (iii) classical microbiological methods that are both qualitative and quantitative. Here we suggest that solar panel biofilms are accessible and highly relevant objects to study microbial ecology, geobiology and biodeterioration.
In this work, we applied scanning electron
microscopy (SEM), microanalysis and Raman spectroscopy
to study the fungi inhabiting a richly illuminated parchment
document and the damage induced by their activity. To that
aim, we collected samples of fungal mycelium from the
deteriorated areas on a removable adhesive tape specifically
intended for lifting fungi without damaging the support.
SEM analysis of the adhesive tape samples showed the cooccurrence
of several species of fungi. One strain closely
resembling Acremonium species was observed only in the
tape micrographs but no agar cultures were obtained. Its
fungal structures showed the production of abundant
oxalates with an outstanding leaching of the calciumbased
materials of parchment (typically manufactured with
gypsum and lime). Needle-like crystals of calcium oxalate
produced by the fungus forming a uniform and quite
regular grid around conidial slimy heads were documented.
As a result, the areas affected by moulds were weakened,
stained and characterised by a powdery patina rich in
calcium. Confocal µ-Raman confirmed the presence of
oxalates while EDS showed the presence of calcium in
crystals. We conclude that the defacement of the parchment
was due to both collagenolytic activity, and to the
biotransformation of calcium-based minerals by fungi.
Due to their ability to penetrate, deteriorate and discolour stone surfaces, rock-inhabiting black fungi represent a remarkable issue for cultural heritage conservation. Black microcolonial fungi (MCF) can also adapt to different environmental conditions, by converting from yeast-like morphology to a peculiar meristematic development with swollen cells (torulose hyphae, TH), to extremely thin structures (filamentous hyphae, FH). Furthermore, black MCF produce protective pigments: melanin, dark pigment particularly evident on light stone surfaces, and carotenoids. Black fungi produce melanin in critical, oligotrophic conditions as well as constitutively. Melanin function is mostly related to stress resistance and the ability of fungi to generate appressorial turgor to actively penetrate plant cells in pathogenic species. An involvement of melanins in stone surface penetration has been suggested, but not experimentally proved. In this work, we tested the role of hyphal melanisation in penetration mechanisms on the model black fungus Knufia petricola A95 in lab conditions. The wild-type and three mutants with introduced targeted mutations of polyketide-synthases (melanin production) and/or phytoene dehydrogenase (carotenoid synthesis) were inoculated on artificial carbonate pellets (pressed Carrara marble powder) of different porosity. After 5, 10, 17 and 27 weeks, hyphal penetration depth and spread were quantified on periodic acid Schiff-stained cross-sections of the pellets, collecting measurements separately for TH and FH. Droplet assay of the mutants on different media were conducted to determine the role of nutrients in the development of different fungal morphologies. In our in vitro study, the hyphal penetration depth, never exceeding 200 μm, was proven to be consistent with observed penetration patterns on stone heritage carbonate substrates. Pellet porosity affected penetration patterns of TH, which developed in voids of the more porous pellets, instead than actively opening new passageways. Oppositely, the thin diameter of FH allowed their penetration independently of substrate porosity. Instead, the long-hypothesized crucial role of melanin in black MCF hyphal penetration should be rejected. TH were developed within the pellets also by melanin deficient strains, and melanized strains showed an endolithic component of non-melanized TH. FH were non-melanized for all the strains, but deeply penetrated all pellet types, with higher penetration depth probably related to their potential exploratory (nutrient-seeking) role, while TH may be more related to a resistance to surface stress factors. In the melanin deficient strains, the absence of melanin caused an increased penetration rate of FH, hypothetically related to an earlier necessity to search for organic nutrients.
Microbiologically influenced corrosion (MIC) is a phenomenon of increasing concern which affects various materials and sectors of society. MIC describes the effects, often negative, that a material can experience due to the presence of microorganisms. Unfortunately, although several research groups and industrial actors worldwide have already addressed MIC, discussions are fragmented, while information sharing and willingness to reach out to other disciplines is limited. A truly interdisciplinary approach, that would be logical for this material/biology/chemistry-related challenge, is rarely taken. In this review we highlight critical non-biological aspects of MIC that can sometimes be overlooked by microbiologists working on MIC but are highly relevant for an overall understanding of this phenomenon. Here, we identify gaps, methods and approaches to help solve MIC related challenges, with an emphasis on the MIC of metals. We also discuss the application of existing tools and approaches for managing MIC and propose ideas to promote an improved understanding of MIC. Furthermore, we highlight areas where the insights and expertise of microbiologists are needed to help progress this field.
Materials are subject to environmental constraints that include biological, chemical and physical factors. To gain confidence about durability and long-term performance of any material, environmental resistance testing procedures have to be amended with modern simulation procedures that include biological components. In fact, any environmentally exposed surface at temperatures lower than 121 °C will be home to microbial growth, even at high salt concentrations, extreme pH, environmental pollution, low water potential, and intense irradiation – everywhere where water is liquid and available. As a consequence, complex microbial ecosystems called biofilms are self-sufficient and found on almost all solid-air-water interfaces. Obviously, environmental changes perturb biofilm development but over a number of seasons, these changes result in relatively stable microbial communities peculiar and adapted to a particular niche and material. Certain microbial settlers are indicative of, and in a real sense mark, a particular biofilm and can, thus, be considered as “reference organisms”. Characteristic reference organisms’ peculiar to specific material-inhabiting communities can be isolated, identified, characterised and used in standard test procedures as well as research into materials science (materials improvement). In this presentation classical microbiological, genetic and molecular methods for studying reference organisms and their roles in materials deterioration will be presented. We will present a set of different reference organisms that are currently in focus of our research and testing development.
Two novel species of the fungal genus Ochroconis, O. lascauxensis and O. anomala have been isolated from the walls of the Lascaux Cave, France. The interest in these fungi and their melanins lies in the formation of black stains on the walls and rock art which threatens the integrity of the paintings. Here we report solid-state cross polarization magic-angle spinning 13C and 15N nuclear magnetic resonance (NMR) spectroscopy and surface-enhanced Raman spectroscopy (SERS) of the melanins extracted from the mycelia of O. lascauxensis and O. anomala in order to known their chemical structure. The melanins from these two species were compared with those from other fungi. The melanins from the Ochroconis species have similar SERS and 13C and 15N NMR spectra. Their chemical structures as suggested by the data are not related to 3,4-dihydroxyphenylalanine, 5,6-dihydroxyindole or 1,8-dihydroxynaphthalene precursors and likely the building blocks from the melanins have to be based on other phenols that react with the N-terminal amino acid of proteins. The analytical pyrolysis of the acid hydrolysed melanin from O. lascauxensis supports this assumption.
Soil formation on weathering rock surfaces is intrinsically connected with the development of primary microbial colonization at the atmosphere-lithosphere interface. A great number and variety of microorganisms is involved in these microbial communities, which are dominated by fungi, algae, cyanobacteria and heterotrophic bacteria. Rock-inhabiting life is ubiquitous on rock surfaces all around the world, but the laws of its establishment, and more important, quantification of its biodeterioration and geological input are possible only in well-controlled and simplified laboratory models. Here we would like to compare two model rock biofilm consisting of the heterotrophic and the phototrophic interacting partners. In the present work the growth of these model biofilms on diverse materials with different physical and chemical properties was investigated under well-controlled laboratory conditions. To clarify the role of environmental factors, the parameters temperature, light intensity and relative humidity were varied in growth test series. For an accelerated substrate colonization and to increase the biomass yield different flow-through chambers systems with semi-continuous cultures have been applied, simulating weathering conditions like flooding, desiccation and nutrient input. The biofilm development was studied by (i) confocal laser scanning and electron microscopy and (ii) qualitatively and quantitatively with respect to cell forms and biomass. A correlation between the presence of the model biofilm and mineral surface alteration as well as geochemical tracers of weathering were followed on various rock substrates (with differing geochemistry, porosity etc) exposed in another flow-through chamber, filled with crushed rock material. Under mentioned environmental conditions different types of flow-through chambers have been used and will be compared.
The biodeterioration of water-based manufactured formulations, such as polymer dispersions, is a major problem for the producing companies and the users of such products. Industrial preservatives, also known as biocides, are therefore used to protect these and similar products from the effects of microorganisms, predominantly bacteria and yeasts. In the absence of internationally recognised standard methods for determining the resistance of polymer dispersions to microbial growth and the efficacy of biocides used in them, protocols for testing other products, e.g., paints, have been adapted, and other methods produced by manufacturing companies, test laboratories, and academic institutions have been used. Often these do not take into account the specific nature of the materials being tested, the types of organism commonly causing contamination, and the conditions that the polymer dispersions will be exposed to during manufacture, storage, and use. By conducting a series of round-robin, collaborative tests, the member organisations of the International Biodeterioration Research Group Polymer Dispersion Working Group have identified the bacteria that commonly infect polymer dispersions, defined the main parameters necessary for a standard method of test, and developed a protocol that is robust, repeatable, and reproducible. The recommended test involves three repeat inoculations of the material with a previously determined mix of seven Gram-negative bacteria and evaluation of living cells by a simple plating technique. The work reported here, carried out by nine participating laboratories, is a final statistical validation and suggests that the method is eminently suitable as a standard test method.