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Background
Spirochetes are the most abundant bacterial group in the hindgut of termites. The largest species, with cell lengths of up to 100 µm, have been provisionally classified in the family “Pillotinaceae” based exclusively on morphological traits. However, in the absence of cultured representatives, their phylogenetic position and metabolism remain entirely unknown.
Results
We investigated phylogeny and metabolic capacities of “pillotinaceous” spirochetes using single-cell techniques, electron microscopy, and fluorescence in situ hybridization. All sequences of large spirochetes obtained from various termites fell into four distinct, well-supported clusters within the family Breznakiellaceae. Based on ultrastructural features, three of the clusters were assigned to the genera Pillotina, Hollandina, and the newly established genus Hollandinoides; a fourth cluster was tentatively assigned to the genus Clevelandina . Functional analysis of the single-cell genomes of Pillotina corrugata sp. nov., Hollandina grandis sp. nov., and Hollandinoides gharagozlouae gen. nov. sp. nov., combined with comparative genomics of other uncultured relatives, demonstrated differences in the capacity to degrade cellulose, hemicelluloses, and dextrins. While members of the genus Pillotina have a fermentative metabolism, members of the other genera encode a Wood–Ljungdahl pathway and, in the case of Hollandina, a group-III nitrogenase, suggesting roles in reductive acetogenesis and nitrogen fixation.
Conclusions
Our results provide the first molecular data on pillotinaceous spirochetes. We show that the three genera covered in our study belong to the family Breznakiellaceae, which harbors the majority of termite-gut spirochetes. Comparative genome analysis indicated that the large spirochetes in termite guts have distinct roles in symbiotic digestion.
Recycling of Li-ion batteries (LiBs) for metal recovery has gained increasing attention in recent years. Batteries contain per- and polyfluoroalkyl substances (PFAS), however, their behaviour during battery recycling is still not well understood. This study aims to (i) characterise the presence of PFAS in LiBs black mass collected from various recycling factories in Australia, and (ii) investigate the fate of PFAS during the metal recovery process. The concentration of bis-perfluoromethanesulfonimide (bis-FMeSI) (C2) in the black mass was up to 51,000 µg kg−1. Other emerging and legacy PFAS were present, with concentrations varying from 0.1 to100 µg kg−1. The complementary analysis results of extractable organically bound fluorine and Fluorine K-edge X-ray adsorption near-edge structure indicates that the LiBs black mass mainly consists of bis-FMeSI (C2) and LiPF6 as the main PFAS analytes (40 – 80% fluorine equivalent), however other unknown PFAS may also be present. The long-chain PFAS are more difficult to leach compared to the short-chain PFAS. H2SO4 leaches 58% bis-FMeSI which is the highest compared to HNO3 (51%) and HCl (40.4%). During the precipitation stage, adding H2O2 to the H2SO4 (5% v/v) leaching agent increased bis-FMeSI adsorption onto metal precipitates by 40%. Using PiFM analysis, PFAS are found predominantly present as surface-associated species within binder- and carbon-rich domains, and the leaching mechanism is strongly attributable to the disruption of these surface-accessible phases. This work constructs the first baseline for the relevant research about the trade-off between metal recovery and PFAS pollutants in the LiBs recycling process.
Microbiologically influenced corrosion (MIC) poses a significant threat to metallic infrastructure across sectors—from energy and marine environments to cultural heritage conservation and emerging technologies such as underground hydrogen storage (UHS). Methanogenic archaea have emerged as key contributors to corrosion under anaerobic conditions, capable of directly interacting with metal surfaces via extracellular electron transfer.
This presentation provides an overview of our recent work on identifying, characterizing, and mitigating MIC caused by methanogens. We focus on the genetic differentiation of methanogenic strains, highlighting a novel class of [Ni/Fe]-hydrogenases identified exclusively in corrosive methanogens to date. These enzymes may serve as molecular markers for MIC risk assessment due to their unique sequence and functional properties.
Additionally, we explore strain-specific differences in hydrogenase glycosylation and correlate these with observed variations in corrosion severity, biofilm formation, microbial surface interactions, and potentially enzyme stability. These findings suggest glycosylation may play a previously underappreciated role in MIC dynamics.
To mitigate MIC, we investigate polyoxometalate-based (POM) coatings—originally developed for protecting stone-based artifacts—which we now apply to metal surfaces. These multifunctional coatings effectively inhibit biofilm formation and microbial activity, offering a promising strategy for corrosion control.
Furthermore, we have developed customized test systems that simulate realistic environmental conditions, including high-pressure settings and dynamic flow regimes with varying velocities. These platforms allow for controlled evaluation of microbial corrosion under conditions relevant to underground hydrogen storage and marine environments, where salinity, pressure, and microbial activity interact.
Our findings demonstrate that integrating molecular diagnostics, surface engineering, and advanced simulation platforms provides new insights into MIC mechanisms and opens avenues for predictive diagnostics and sustainable corrosion control strategies in industrial applications.
Mikrobielle Einflüsse auf Wasserstoffspeicherung: Materialien, Abbauprozesse und Teststrategien
(2026)
Wasserstoff gilt als Schlüsseltechnologie für die Energiewende – doch seine sichere Anwendung stellt neue Anforderungen an Technik, Infrastruktur und Personal. Die Veranstaltung „H₂ Sicherheit“ bietet eine umfassende Plattform, um sich über die sicherheitsrelevanten Aspekte von Wasserstoff zu informieren und praxisnahe Lösungen kennenzulernen. Expert:innen aus Forschung, Industrie und Netzbetrieb geben Einblicke in aktuelle Entwicklungen, Herausforderungen und Best Practices. Fokus dieser Präsentation war der Mikrobielle Einfluss bei der unterirdischen Speicherung von Wasserstoff und das neuartige Testsystem (MISTRAL)
BPA unterliegt einer kontinuierlichen behördlichen Kontrolle und ist als besonders besorgniserregender Stoff (SVHC) gelistet. Besondere Besorgnis besteht hinsichtlich seines häufigen Nachweises in Oberflächengewässern, obwohl es leicht biologisch abbaubar ist. In mehreren Studien wurden die Quellen und Wege von BPA in die Umwelt untersucht, wobei festgestellt wurde, dass seine Hauptverwendung als Monomer in PC nur marginal zu den BPA-Freisetzungen in die Umwelt über seinen Lebenszyklus beiträgt.
Um die tatsächlichen Freisetzungen von BPA aus PC unter umweltrelevanten Bedingungen besser zu verstehen, wurde eine neu entwickelte Methodik (Bundesanstalt für Materialforschung und -prüfung, BAM) angewendet, die ein neuartiges beschleunigtes Bewitterungsprotokoll für Polycarbonat-Materialien in Kombination mit einem sensitiven Analysenverfahren umfasst, welches eine Bestimmung von BPA im Ultraspurenbereich ermöglicht. Das Bewitterungsprotokoll erreicht eine 13,6-fache Beschleunigung im Vergleich zu mitteleuropäischen Wetterbedingungen und simuliert Umweltstressoren (globale Strahlung, Regen, Temperaturschwankungen) in einer Bewitterungskammer, wobei gleichzeitig die BPA-Freisetzung mit einem validierten LC-MS/MS-Verfahren unter Verwendung einer organischen Isotopenverdünnungskalibrierung bestimmt wird. In parallelen Freilandversuchen wurden die Trübung und die Vergilbung („Yellowness-Index“) der zu untersuchenden Proben als Referenzparameter untersucht.
BPA unterliegt einer kontinuierlichen behördlichen Kontrolle und ist als besonders besorgniserregender Stoff (SVHC) gelistet. Besondere Besorgnis besteht hinsichtlich seines häufigen Nachweises in Oberflächengewässern, obwohl es leicht biologisch abbaubar ist. In mehreren Studien wurden die Quellen und Wege von BPA in die Umwelt untersucht, wobei festgestellt wurde, dass seine Hauptverwendung als Monomer in PC nur marginal zu den BPA-Freisetzungen in die Umwelt über seinen Lebenszyklus beiträgt.
Um die tatsächlichen Freisetzungen von BPA aus PC unter umweltrelevanten Bedingungen besser zu verstehen, wurde eine neu entwickelte Methodik (Bundesanstalt für Materialforschung und -prüfung, BAM) angewendet, die ein neuartiges beschleunigtes Bewitterungsprotokoll für Polycarbonat-Materialien in Kombination mit einem sensitiven Analysenverfahren umfasst, welches eine Bestimmung von BPA im Ultraspurenbereich ermöglicht. Das Bewitterungsprotokoll erreicht eine 13,6-fache Beschleunigung im Vergleich zu mitteleuropäischen Wetterbedingungen und simuliert Umweltstressoren (globale Strahlung, Regen, Temperaturschwankungen) in einer Bewitterungskammer, wobei gleichzeitig die BPA-Freisetzung mit einem validierten LC-MS/MS-Verfahren unter Verwendung einer organischen Isotopenverdünnungskalibrierung bestimmt wird. In parallelen Freilandversuchen wurden die Trübung und die Vergilbung („Yellowness-Index“) der zu untersuchenden Proben als Referenzparameter untersucht.
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.