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Black fungi are a polyphyletic group of melanized ascomycetes adapted to extreme environments such as deserts, rocks, and human-made surfaces. Their slow compact growth, and thick melanized cell walls confer exceptional stress tolerance but have long hindered molecular studies and genetic engineering. Consequently, the biology and biotechnological potential of these organisms remained largely unexplored. To overcome these limitations, we selected the rock-inhabiting black fungus Knufia petricola as a model. Its genome sequence (12 contigs, ~10,000 genes) enabled the development of a versatile genetic toolbox based on CRISPR/Cas9-mediated editing, achieving up to 100% homologous recombination and supporting multiplex editing of at least six genomic regions. Color-based selection systems targeting pigment genes enable rapid identification of strains with correctly integrated expression cassettes, including simultaneous insertion of optimized genes coding for blue, green, and red fluorescent proteins for co-localization and protein interaction studies. These tools enable functional analyses of traits central to extremotolerance, such as the role of melanin in stress protection, biofilm formation, and material interactions. Furthermore, K. petricola has been established as a eukaryotic expression platform: heterologous genes from bacteria, fungi, and plants can be expressed under constitutive or inducible promoters, including the Tet-on system for tunable gene regulation. Pigment-free strains provide access to metabolic precursors, enabling high-level synthesis of alternative secondary metabolites. Importantly, the optimized transformation protocols were successfully transferred to the Antarctic black fungus Cryomyces antarcticus, a cryptoendolithic extremophile and astrobiology model, demonstrating that even the most stress-tolerant fungi can be genetically engineered. Together, the genome sequence and advanced genetic engineering strategies have transformed K. petricola from a challenging extremotolerant organism into a tractable model for studying stress adaptation, material interactions and a promising host for biotechnology and synthetic biology approaches. These achievements, combined with emerging black fungal genomes, inform sustainable material protection strategies to mitigate colonization and biodeterioration of exposed surfaces, linking fundamental research with applied solutions for material conservation and industrial biotechnology.
Iron is an essential micronutrient for all organisms, driving intense competition for its acquisition. This competition is particularly evident in parasitic interactions, where hosts actively restrict iron availability to inhibit the growth of fungal invaders (nutritional immunity). Saprobic fungi, on the other hand, acquire iron by digesting dead organic material and may protect their nutrient sources from competitors through efficient iron uptake, rapid growth, and the production of toxic secondary metabolites. Rock-inhabiting black fungi colonize – alone or within microbial communities (subaerial biofilms) – oligotrophic, exposed surfaces such as rocks, stone monuments, and photovoltaic panels, where competition from fast-growing saprobes is minimal. To understand how these fungi acquire iron, we combined comparative genomics with targeted gene deletions in the model species Knufia petricola. Genome analysis revealed a reduced repertoire of iron acquisition-related genes compared to those of pathogenic relatives: a single reductive iron assimilation (RIA) complex, one non-ribosomal peptide synthetase (NRPS) for siderophore biosynthesis, and absence of vacuolar iron transporters. Functional assays demonstrated that K. petricola primarily relies on RIA, mediated by FTR1–FET1, and secondarily on siderophore-mediated iron acquisition (SIA) via NPS1. Mutants lacking both systems were nearly non-viable under iron limitation, confirming that RIA and SIA are the only relevant uptake routes under laboratory conditions, that simulate the free-living lifestyle. Cross-feeding and chemotropism assays indicated secretion of an extracellular siderophore enabling growth toward the iron-containing mineral olivine. This siderophore, however, failed to mobilize iron from strong chelators such as BPS and EDTA, and its chemical nature remains unknown. Although melanin adsorbs and reduces iron, results on deletion mutants showed that melanin does not contribute to iron uptake or chelator resistance, functioning instead as a passive iron sink. Sensitivity assays revealed unusually low minimal inhibitory concentrations (MICs) for these chelators in K. petricola and other rock-inhabiting fungi, suggesting limited iron acquisition and storage capacity. These findings highlight the vulnerability of rock-inhabiting fungi to iron depletion and suggest that strong iron chelators could serve as an effective strategy to inhibit fungal colonization of human-made surfaces.
Black fungi, a diverse group of ascomycetes, well known for their ability to thrive in extreme environments, are found to colonize and grow on subaerial surfaces where few other microbes survive. This includes bare rock as well as marble monuments and solar panels. Black fungi can deteriorate hard substrates which is of particular concern for the preservation of cultural heritage, while their ability to colonize solar panels can cause a reduction in green energy yield. We have been developing computational modelling tools to gain predictive insight into their growth and proliferation. In this work we focus on the black fungus Knufia petricola, a fungus that possesses many of the characteristic features of black fungi generally and for which genetic engineering tools have been developed. Detailed observations and systematic characterization of the organism are fundamental to developing a predictive model. We combined the lessons from individual-based and biofilm characterization studies of K. petricola by Dehkohneh et al. (Manuscripts in preparation) with the predictive potential of Individual-based Modelling (IbM). In these systematic characterization studies K. petricola was grown and characterized on agar and on a glass substrate. K. petricola was grown under a variety of conditions; different carbon and nitrogen sources, different degrees of nutrient limitation, and C:N ratios. We"ve thus gained insight on how these conditions affect the growth characteristics and morphological development of K. petricola, including how nutrient limitation can limit branch development and shift generation times, and how the C:N ratio affects substrate penetration. We employed this knowledge to develop a new K. petricola IbM.We used the IbM framework iDynoMiCS 2.0 (Cockx et al. 2024) to formulate our model. The model captures both morphological and kinetic aspects of K. petricola. By formulating characteristics of individual cells and the interactions with their neighbors and environment mathematically we can simulate emergent properties, such as the architecture and expansion of K. petricola micro- colonies, which gives us the unique ability to link microbial traits with biofilm characteristics. The model gives us insight on how environmental parameters can be manipulated to steer the development of K. petricola biofilms, it can thus help forecast K. petricola biofilm proliferation and can be used to inform new bio-colonization mitigation strategies.
Microcolonial black fungi, commonly found on sun-exposed natural and man-made surfaces worldwide, belong to different classes within the Ascomycota but convergently evolved similar morpho-physiological adaptations to colonize extreme low-competitive environments. Genetic studies of these organisms have long been hampered by slow growth, lack of sexual cycles and difficulties in transformation. To overcome these limitations, CRISPR/Cas9-based genome editing was implemented in the rock-inhabiting fungus Knufia petricola (Eurotiomycetes, Chaetothyriales). This enables efficient generation of deletion mutants and overexpression strains for functional analyses, and thus hypothesis-driven targeted mutagenesis. However, the unique ability of black fungi to colonize oligotrophic extreme environments remains poorly understood, and genes involved are unknown. To address this, a hypothesis-generating tool for functional assessment of new species- and/or trait-specific genes was implemented in K. petricola. Specifically, we adapted the two-component Activator/Dissociation (Ac/Ds) transposon system from maize for generating insertional mutants by in vivo mutagenesis. For controlling the transposition of a Ds transposon carrying a resistance cassette, the inducible and metabolism-independent Tet-on promoter system was combined with the Ac transposase (AcTPase) coding sequence fused to a functional nuclear localization signal. In total, six auxotrophic Ac/Ds starter strains were generated, each harboring the Ds transposon at different positions within ade2, ura3 or ppt1. Induction of TET::AcTPase with doxycycline followed by selection on ADE/URA/LYS-lacking media resulted in prototrophic revertants for most Ac/Ds strains. Sequencing of excision sites revealed characteristic footprints. Mapping of Ds re-insertion sites demonstrated transpositions both within the same chromosome and across different chromosomes, identifying 30 genes as non-essential. Current efforts include scaling up mutant generation, as the generation and sequencing of saturated mutant libraries combined with mutation mapping will enable the systematic identification of essential genes under diverse culture conditions.
Sun-exposed surfaces are extreme environments characterized by intense UV radiation, temperature fluctuations, desiccation, and nutrient scarcity. These habitats are colonized by extremotolerant microorganisms that often form multispecies biofilms. Among them, black fungi (Ascomycota) stand out for their unique morphophysiological traits: slow growth, small compact colonies, reproduction by budding or meristematic growth, and continuous deposition of 1,8-dihydroxynaphthalene (DHN) melanin on thickened cell walls. This melanin is thought to provide protection against abiotic stress, for both the fungi and their microbial partners. Originally associated with natural substrates such as rocks in hot, cold, or saline deserts, black fungi have recently been detected on human-made surfaces, including solar panels, where they may reduce system efficiency. Despite their simple morphology, black fungi represent a vast and underestimated genetic diversity with representatives in Eurotiomycetes, Dothideomycetes, and Arthoniomycetes. They exhibit diverse lifestyles, including lichen symbioses, opportunistic pathogenicity, and saprobic growth. However, only a few genomes of black fungi have been sequenced to date. Initial analyses indicate haploid or diploid states, occasional genome duplications, and gene loss, reflecting adaptation to extreme conditions. For even fewer species, genetic engineering tools are available. To address this gap, we develop genetic tools for functional studies and compare genome sequences. The STRES Community Science Program, funded by the Joint Genome Institute (JGI), aims to sequence up to 600 black fungal genomes from diverse extreme environments (https://stresblackfungi.org/). Our contribution includes black fungi isolated from solar panels in Germany and the United States – among some several new species. Annotated genomes are now becoming available, enabling comparative analyses to discover novel genes for enzymes and secondary metabolites. These efforts also support strategies to mitigate undesired biofilm formation on man-made surfaces such as facades and solar panels.
The climate crisis is driving an increasing demand for ecologically oriented concepts. In the building sector, this demand includes not only the use of environmentally friendly materials but also the greening of urban areas. One promising approach is the development of bioreceptive concrete façades, which support the growth of green biofilms directly on their surfaces. These innovative façades are anticipated to deliver benefits comparable to those of macroscopically greened façades, such as enhanced biodiversity and improved air quality, while offering the advantages of being more self-sustaining and stable systems once fully established.
However, the development of bioreceptive concrete presents substantial challenges. Due to the interdisciplinarity and novelty of this field, standardized methods for material characterization and bioreceptivity assessment are currently lacking. This study proposes an approach for evaluating surface properties crucial for bioreceptivity, developed on differently structured samples of ultra-high-performance concrete (UHPC). Existing methods and standards from concrete technology are critically reviewed and, where necessary, modified to meet the unique requirements of measuring bioreceptive material properties. Special attention is given to the surface pH value and water retention characteristics, as these are essential for promoting microbial growth and ensuring the long-term stability of green biofilms. The observed surface characteristics vary according to the imprinted surface structures, offering a spectrum of material properties and enabling the evaluation of their impact on bioreceptivity. The findings presented form the foundation for subsequent laboratory weathering experiments, which will be discussed in a complementary publication.
A 30-year-old diesel tank: Fungal-dominated biofilms cause local corrosion of galvanised steel
(2026)
The increased use of biodiesel is expected to lead to more microbial corrosion, fouling and fuel degradation issues. In this context, we have analysed the metal, fuel and microbiology of a fouled diesel tank which had been in service for over 30 years. The fuel itself, a B7 biodiesel blend, was not degraded, and—although no free water phase was visible—contained a water content of ~60 ppm. The microbial community was dominated by the fungus Amorphotheca resinae, which formed thick, patchy biofilms on the tank bottom and walls. The tank sheets, composed of galvanised carbon steel, were locally corroded underneath the biofilms, up to a depth of a third of the sheet thickness. On the biofilm-free surfaces, Zn coatings could still be observed. Taken together, A. resinae was shown to thrive in these water-poor conditions, likely enhancing corrosion through the removal of the protective Zn coatings.
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.
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.
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.