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Eingeladener Vortrag
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Fungal pigments such as melanin and carotenoids are distinctive markers of animal and plant pathogenic fungi as well as their environmental relatives. These complex pigments play important roles in pathogenicity and stress tolerance while also being useful as biomarkers. Accordingly, it is important to be able to identify in situ the pigments in black fungi, a group of clinical and environmental importance. In this study, wild-type and genetically modified strains of Knufia petricola A95 and wild fungal cells attached to ancient rock were investigated for their spectroscopic and microscopic Raman features and morphological appearance. Knockout mutants of melanin synthesis genes pks1 (polyketide synthase), sdh1 (scytalone dehydratase), and both pks1 and the carotenoid synthesis gene phd1 (phytoene desaturase) were studied We applied two different Raman microscopes using two lasers, with 633 nm and 488 nm wavelengths. We analyzed and compared Raman spectra between the measured reference substances and the mutant and wild-type strains. In the wild strain WT:A95, the peaks close to melanin peals were found at 1353 cm−1 and 1611 cm−1. There are no characteristic melanin peaks at 1580–1600 cm−1 and around 1350 cm−1 at the spectrum of the Δpks1/Δphd1 mutant and the Δsdh1 mutant. The Δpks1 mutant spectrum has the peaks at the beta-carotene v2 C-C in-plane stretch at 1155 cm−1 and v3 C-CH3 deformation at 1005 cm−1. The peaks of carotenoids and melanin were found in all mutants and the wild strain, except the Δpks1/Δphd1 mutant. Raman spectra allow for discrimination between the various pigments. Hence, interactions between natural fungal melanin, as well as other protective pigments, and complex environmental matrices can be characterized on a range of spatial and temporal scales.
Human-made systems, also called “build environment” or “technosphere”, sustain human comfort as well as our industrial activities. These systems have become particularly widespread since the Industrial Revolution, i.e., since the 17th century. At the same time, these technical systems – buildings, monuments, energy production, transformation and transmission, water purification and supply systems - serve as new habitats for living organisms.
Life is ubiquitously present on our planet since a very long time: the Earth is 4.54 billion years old and microbial communities have played a key role on our planet for 3.7 billion years. Once human-made system appeared, microorganisms became an integral part of all types of technosphere infrastructure as well.
Here we will illustrate biosphere-technosphere interactions using a specific example of the black fungi and their impact on the efficiency of solar (photovoltaic) panels. This expanding renewable infrastructure for electricity generation is growing on all continents - and create a specific, arid habitat for stress-tolerant black fungi.
Black fungi were once discovered in hot and cold natural deserts – and now belong to the persistent colonisers of human-made deserts of solar parks. This new niche is evolving an impressive biodiversity. So far more than 60 isolates of black fungi belonging to Arthoniomycetes, Eurotiomycetes and Dothideomycetes were obtained from solar panels in Europe and Americas. Here we will present the analysis of this emerging anthropogenic biodiversity.
Opportunities for future research in the field include quantification of the microbial load on technosphere surfaces – along with characterisation of the corresponding microbial diversity. The strategy of precise measurement and characterisation will enable us to reliably determine the beneficial and harmful functions that living microorganisms play in the functioning of energy-generating systems – and technosphere in general.
Nutrient acquisition strategies of plants regulate water flow and mass transport within ecosystems, shaping earth surface processes. Understanding plant strategies under current conditions is important to assess and predict responses of natural ecosystems to future climate and environmental changes. Nitrogen (N) and potassium (K) (re-)utilization from topsoil and their acquisition from subsoil and saprolite were evaluated in a continental transect, encompassing three study sites – an arid shrubland, a mediterranean woodland, and a temperate rainforest – on similar granitoid parent material in the Chilean Coastal Cordillera. The short-term (<1 year) plant N and K acquisition was traced with 15N and the K analogs rubidium and cesium. To do so, the tracers were either injected into topsoil, subsoil, or saprolite, in the immediate vicinity of eight individual plants per study site and injection depth. The long-term (>decades) K uplift by plants was investigated by the vertical distribution of exchangeable K+ and Na+. Recoveries of 15N and K analogs by arid shrubland plants were similar from topsoil, subsoil, and saprolite. Mediterranean woodland shrubs recovered the tracers primarily from topsoil (i.e., 89 % of recovered 15N and 84 % of recovered K analogs). Forest plants recovered the tracers from topsoil (15N = 49 %, K analogs = 57 %) and partially from greater depth: 38 % of recovered 15N and 43 % of recovered K analogs were acquired from subsoil and saprolite, respectively. Low nutrient accessibility in the topsoil (e.g., because of frequent droughts) drives shrubland plants to expand their N and K uptake to deeper and moister soil and saprolite. Woodland and forest plants dominantly recycled nutrients from topsoil. In the forest, this strategy was complemented by short-term uplift of N and K from depth. The vertical distribution of exchangeable K indicated long-term uplift of K by roots in all three sites. This highlighted that long-term K uplift from depth complements the nutrient budget across the continental transect.
Drei grundlegend verschiedene Ansätze für Umweltsimulation werden an Beispielen illustriert:
(i) Ganzheitlicher Ansatz - Nachstellen von Umweltmilieus im Labor
Ziel ist hier das Nachstellen von (kombinierten) Umweltbedingungen im Labor; die Umweltparameter werden mit all ihren Wechselwirkungen aufgebracht.
Hauptnutzen ist eine gegenüber der natürlichen Beanspruchung erhöhte Reproduzierbarkeit der Umweltbedingungen. Hat man sein Laborsetup entwickelt, ist es auf verschiedene Materialien anwendbar.
Unter solchen Laborbedingungen ermittelte Lebensdauern sind dabei nicht auf die typischerweise sehr variablen Real-Umweltbedingungen übertragbar.
(ii) Parametrisierter Ansatz - Ermittlung einzelner Materialempfindlichkeiten
Hierbei werden im Labor die Wirkungen separater Umweltparameter auf Materialien nachgestellt. Für eine solche Separation der Einflussfaktoren ist insbesondere die Aufschlüsselung möglicher Wechselwirkungen der Umwelt-parameter (z.B. Mikroklima an bestrahlten Oberflächen) erforderlich. Einzelne (meist Alterungs-) Empfindlichkeiten können qualitativ nachgewiesen werden oder sogar – als Beanspruchungs-Wirkungs-Funktionen – quantifiziert werden, was einen wesentlichen Schritt in Richtung der Digitalisierung der Material¬prüfung darstellt. Insbesondere ist dann auch eine Lebensdauer-vorhersage für vorgegebene Zeitreihen der Beanspruchungs¬parameter umsetzbar.
(iii) Rückwirkungen auf die Umwelt
Umweltbeanspruchungen können zur Freisetzung von Schadstoffen in die Umwelt führen. Durch die Nachstellung kritischer, aber realitätsnaher Einsatzszenarien kann die Menge an freigesetzten Substanzen abgeschätzt werden.
Egal, welcher Ansatz verfolgt wird – ein Vergleich mit der oder einer Real-beanspruchung ist unerlässlich, ebenso wie die Messdatenaufzeichnung (data logging) aller potenziell relevanten Beanspruchungsparameter während dieser Realbeanspruchung.
Obwohl die naturnahe Umwelt – sowohl in der BAM als auch bei der GUS – gegenüber der technischen Umwelt eher untergeordnet auftritt, werden zur Illustration Beispiele aus der naturnahen Umwelt verwendet.
Vielleicht nannten es die Berliner „Puppen putzen“, wenn in den 1880er Jahren die Marmorstatuen auf der Schlossbrücke vor dem Berliner Stadtschloss mit Bürste, Wasser und Seife von Staub, Ruß und Taubendreck befreit wurden. Soviel Pflege wurde diesen Göttinnen aus der griechischen Mythologie nicht immer zuteil. So haben Klima, Luftverschmutzung und der 2. Weltkrieg, wie bei anderen Kunstwerken auch, tiefe Spuren hinterlassen. Der Erhalt dieser zwischen 1847 und 1857 aufgestellten Skulpturen und vieler vergleichbarer Objekte im Stadtbild ist durch die auch heute weiter fortschreitende Schädigung des Marmors gefährdet. In den Jahren 2006 bis 2010 förderte die Deutsche Bundesstiftung Umwelt (DBU) ein Projekt zur „Entwicklung und Überprüfung von Einhausungssystemen zur Reduzierung umweltbedingter Schädigungen von außenexponierten Marmorobjekten mit dem Ziel des langfristigen Erhalts in situ an einem national bedeutenden Objektkomplex, den Schlossbrückenfiguren unter den Linden, Berlin“. Geleitet und restauratorisch betreut wurde das Projekt durch das Landesdenkmalamt (LDA) Berlin. Durch die Abteilung Strukturgeologie und Geodynamik (GZG) der Georg-August Universität Göttinnen wurden die Verwitterungsmechanismen des Marmors untersucht und auf dieser Basis Anforderungen an das Schutzsystem formuliert. Anhand von numerischen Simulationen des Instituts für Bauklimatik der Technischen Universität Dresden (TUD) wurde das bauphysikalische Konzept des Einhausungssystems optimiert. Die konstruktive Gestaltung eines Prototyps und des finalen Entwurfs erfolgte durch das Fachgebiet Konstruktives Entwerfen und Tragwerkslehre der Universität der Künste Berlin. Durch den Fachbereich Baustoffe der BAM erfolgten die Realisierung des Prototyps der Einhausung sowie ein umfangreiches Klimamonitoring an den Skulpturen. Anhand dieses interdisziplinären Projektes und ergänzt durch weitere Messungen wird gezeigt, mit welchen Methoden die für die Degradation von Marmor relevanten Mechanismen untersucht werden können. Die Erkenntnisse aus dem Projekt sind die Grundlage für die Entwicklung nachhaltiger Pflege- und Schutzkonzepte für die kulturhistorisch wertvollen Skulpturen. Nur mit solchen modernen konservatorischen Programmen können die Originalskulpturen im Berliner Stadtraum erlebbar bleiben.
Plant resource acquisition strategies were ecosystem-specific with distinct mutualism with arbuscular mycorrhizal (AM) fungi. Root traits indicated conservative resource economics in the arid shrubland, but an acquisitive and self-sufficient (“do-it-yourself”) acquisition strategy in the semiarid coastal matorral, resulting in large carbon (C) investments (green). Forest plants with conservative root traits seem to intensively outsource their acquisition to AM fungi, compensating for lower uptake capacities of conservative roots (red line). High allocations of freshly assimilated C into AM fungal storage compounds illustrated the relevance of AM fungi as C sink, especially in the semiarid matorral.
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.
Stress is a normal part of life for fungi, which can survive in environments considered inhospitable or hostile for other organisms. Due to the ability of fungi to respond to, survive in, and transform the environment, even under severe stresses, many researchers are exploring the mechanisms that enable fungi to adapt to stress. The International Symposium on Fungal Stress (ISFUS) brings together leading scientists from around the world who research fungal stress. This article discusses presentations given at the third ISFUS, held in Sao Jose dos Campos, Sao Paulo, Brazil in 2019, thereby summarizing the state-of-the-art knowledge on fungal stress, a field that includes microbiology, agriculture, ecology, biotechnology, medicine, and astrobiology.
Fungi that share light-flooded habitats with phototrophs may profit from their excess photosynthetic products. But to cope with sunlight-associated stresses [e.g. high temperatures, UV radiation with associated DNA damage, accumulation of reactive oxygen species (ROS), desiccation and osmotic stresses] it is important for fungi to accurately sense and respond to changes in light. To test the hypothesis that light is an environmental cue that Ascomycota use to coordinate growth, stress responses as well as to establish pathogenic or symbiotic relationships, the photoreceptor (PR) distribution in species from different ecological niches was analysed. The genomes of black [dihydroxynaphthalene (DHN) melanin-containing] fungi from phyllosphere and exposed solid surfaces contain multiple photoreceptors (PRs). The plant pathogen Botrytis cinerea (Leotiomycetes) has a highly sophisticated photosensory and signalling system that helps to avoid light and to locate susceptible hosts. Rock-inhabiting Dothideomycetes and Eurotiomycetes including Knufia petricola possess equal numbers ofPRs along with the same set of protective pigments. This similarity between black fungi from plant and rock surfaces suggests that photoperception and -regulation are important for fungi that receive nutrients through cooperation with phototrophs. Genetic tools for manipulating K. petricola exist and will be used to test this idea.
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.
After their discovery on rock surfaces in cold and hot deserts, a polyphyletic group of ascomycetous black fungi was found to dominate a range of hostile environments – natural and man-made, from salterns to dishwashers, roofs and solar panels. Together with bacteria and algae they may establish subaerial biofilms and cause weathering of the surfaces they grow on. Their impressive survival abilities as well as their constitutive protective pigmentation and cluster-like microcolony organisation are similar in environmental isolates as well as in heat-tolerant opportunistic pathogens of animals and humans. We selected the rock-inhabiting fungus Knufia petricola (class Eurotiomycetes, order Chaetothyriales) that exhibits all the characteristics of microcolonial black fungi such as yeast-like cell growth, absence of reproductive structures and constitutive dihydroxynaphthalene (DHN) melanogenesis (Nai et al. 2013, Fungal Genet Biol). We developed protocols to efficiently generate and transform protoplasts resulting in stable homokaryotic transformants by targeting genes involved in pigment synthesis. The differences between the wild-type fungus and its melanin-deficient mutants were tested in geomicrobiological experiments and measured are now discussed in detail, with special accent on the possible effects of the mutation on EPS and other exuded substances.
A genetic toolbox for exploring the life style of the rock-inhabiting black fungus Knufia petricola
(2020)
After their discovery on rock surfaces in cold and hot deserts, a polyphyletic group of ascomycetous black fungi was found to dominate a range of hostile environments – natural and man-made, from salterns to dishwashers, roofs and solar panels. Together with bacteria and algae they may establish subaerial biofilms and cause weathering of the surfaces they grow on. Their impressive survival abilities as well as their constitutive protective pigmentation and cluster-like microcolony organisation are similar in environmental isolates as well as in heat-tolerant opportunistic pathogens of animals and humans. The exact genetic properties that ensure their survival in extreme environments can be studied if some black fungi were amenable to genetic manipulations. We selected the rock-inhabiting fungus Knufia petricola (class Eurotiomycetes, order Chaetothyriales) that grows moderately in axenic culture and exhibits all the characteristics of microcolonial black fungi such as yeast-like cell growth, absence of reproductive structures and constitutive dihydroxynaphthalene (DHN) melanogenesis (Nai et al. 2013, Fungal Genet Biol). We developed protocols to efficiently generate and transform protoplasts resulting in stable homokaryotic transformants by targeting genes involved in pigment synthesis and expressing fluorescent reporter genes. Hence, endogenous and foreign genes can be expressed from episomal AMA1-containing plasmids and genome-integrated DNA constructs. Moderate rates of homologous recombination allow for both ectopic and targeted integrations. CRISPR-Cas9 was further validated as a strategy for obtaining selection marker-free mutants and silencing via RNA interference as an approach to study essential genes. Availability of this genetic toolbox and an annotated genome sequence of the strain A95 is paving the way for studying interactions of K. petricola with environmental stressors, material surfaces, soil matrices and phototrophic symbionts.
Low-molecular-weight organic acids (LMWOAs) are crucial for the mobilization and acquisition of mineral phosphorus by plants. However, the role of LMWOAs in mobilizing organic phosphorus, which is the predominant phosphorus form in at least half of the world’s ecosystems, especially in humid climates, is unclear. The mechanisms of phosphorus mobilization by LMWOAs depend on climate, mainly precipitation, and shape the phosphorus nutrition strategies of plants. We disentangled the impact of roots and associated microorganisms on mechanisms of phosphorus cycling mediated by LMWOAs by studying soils along an ecosystem-sequence (ecosequence) from arid shrubland (~70 mm yr-1), and Mediterranean woodland (~370 mm yr-1) to humid-temperate forest (~1470 mm yr-1). Phosphorus speciation in soil was examined by X-ray absorption near edge structure analysis (XANES). LMWOAs were quantified as biological rock-weathering and organic phosphorus mobilization agents and compared to kinetics of acid phosphatase as a proxy for organic phosphorus mineralization. Calcium-bound phosphorus in topsoils decreased from 126 mg kg-1 in the arid shrubland, to 19 mg kg-1 in the Mediterranean woodland and was undetectable in the humid-temperate forest. In contrast, organic phosphorus in topsoils in close root proximity (0–2 mm distance to roots) was absent in the arid shrubland but raised to 220 mg kg-1 in the Mediterranean woodland and to 291 mg kg-1 in the humid-temperate forest. The organic phosphorus content in topsoils was 1.6 to 2.4 times higher in close root proximity (0–2 mm distance to roots) compared to bulk soil (4–6 mm distance to roots) in the Mediterranean woodland and humid-temperate forest, showing intensive phosphorus bioaccumulation in the rhizosphere. Redundancy analysis (RDA) revealed that LMWOAs were explained by the content of hydroxyapatite and variscite phosphorus-species in the arid shrubland, indicating that LMWOAs contribute to mineral weathering in this soil. LMWOA contents, phosphatase activity, and microbial biomass carbon correlated strongly with organic phosphorus in the humid-temperate forest soil, which implies a high relevance of LMWOAs for organic phosphorus recycling. In the Mediterranean woodland soil, however, oxalic acid correlated with organic phosphorus in the topsoil (suggesting phosphorus recycling), whereas in the subsoil malic and citric acid were correlated with primary and secondary phosphorus minerals (implying mineral weathering). We conclude that phosphorus acquisition and cycling depend strongly on climate and that the functions of LMWOAs in the rhizosphere change fundamentally along the precipitation gradient. In the arid shrubland LMWOAs facilitate biochemical weathering (rock eating), while in the humid-temperate forest their functions change towards supporting organic phosphorus recycling (vegetarian).
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.
In the modern world there is an increased understanding that design and performance monitoring of materials have to be tested in connection to chemical, physical and (micro)biological challenges. A systematic study on how biofilms interact with materials and what could be done to engineer biofilms and/or materials in order to maximize the resistance of the material (surface) or the resistance the biofilm-modified material (bulk) is in strong need. In the Department “Materials and the Environment” of the BAM new experimental platform is being developed. With the help of different type of device for high throughput and microbiologically-controlled environment simulation we establish a new approach to clarify the mechanisms of biofilm/material interactions. Despite the focus on fundamental research, the main results of this project proposal will be transferable into material technology and construction chemistry and will influence the development of standardization in this topic. As the interactions of biofilms and materials have implications for most constructions as well as climate change, the results of the research generates additional value.
Interface between the atmosphere and mineral substrates is the oldest terrestrial habitat. Morphologically simple microbial biofilms were the first settlers on these inhospitable surfaces at times when the Earth was inhabited only by microorganisms and the solid substrates represented only by natural rock surfaces i.e. lithosphere. Miniature, self-sufficient microbial ecosystems continue to develop on subaerial (i.e. air-exposed) solid surfaces at all altitudes and latitudes where direct contact with the atmosphere and solar radiation occurs – on rocks, mountains, buildings, monuments, solar panels. All these sub-aerial biofilms develop under fluctuating and hostile conditions – and thus frequently harbour stress-tolerant black fungi inherently able to cope with the stresses of bright sunlight and constantly changing atmospheric conditions. Black fungi – a polyphyletic group of Ascomycetes– accumulate the dark pigment DHN melanin, diverse carotenoids and mycosporines in their cells and thus successfully colonise sunlight-flooded habitats from phyllosphere to rock surfaces. Various chemical and physical extremes and fluctuating environments belong to the challenges effectively mastered by black fungi. In our laboratory we isolate novel black fungi from man-made habitats like building materials and solar panels. Using Knufia petricola A95 as a model we conduct experiments to clarify interactions of black fungi with inorganic substrates. We use available mutants to determine the functional consequences of changes in the outer cell wall envelopes – from excreted EPS to layers of protective pigments. A genetic toolbox to manipulate this Chaetothyriales representative is in further development. Our long-term goal is to understand the fundamental mechanisms how black fungi are able (i) to adhere to dry atmosphere-exposed surfaces, (ii) to survive multiple stresses and (iii) to change the underlying substrates including rocks.
The story of how black fungi survive harsh conditions on sun-exposed desert rocks and material surfaces is fascinating. In the presentation examples of how knowledge of these organisms can be of practical value (e.g., in biodeterioration studies, such as on stone markers in cemeteries) would be given. Among other stories, the example of how roof tiles amended with a titanium oxide layers as a biocide actually selected for the black fungi will be told. A hypothesis about how these organisms would likely be found on solar panels and some early BAM work in that area will be presented. This then brings the connection to a study ripe for investigation in North Carolina. The talk presents the development of a study aiming at deciphering the influence of microbial biofilm formation on the energy conversion efficiency of solar photovoltaic panels or modules at two facilities (one facility under the impact of high intensity of animal agriculture and high deposition of ammonia from atmosphere and the other with low animal agriculture and lower atmospheric ammonia deposition) in North Carolina. The main hypothesis of the study is that microbial biofilm formation on solar photovoltaic panels will lead to significant decreases in energy conversion efficiency of solar photovoltaic modules and biofilm formation will also be accelerated by high ammonia concentration in the ambient atmosphere and high nitrogen deposition.
A Genetic Toolbox for Exploring the Life Style of the Rock-inhabiting Black Fungus Knufia petricola
(2019)
After their discovery on rock surfaces in cold and hot deserts, a polyphyletic group of ascomycetous black fungi was found to dominate a range of hostile environments – natural and man-made, from salterns to dishwashers, roofs and solar panels. Together with bacteria and algae they may establish sub-aerial biofilms and cause weathering of the surfaces they grow on. Their impressive survival abilities as well as their constitutive protective pigmentation and cluster-like microcolony organisation are similar in environmental isolates as well as in heat-tolerant opportunistic pathogens of animals and humans. The exact genetic properties that ensure their survival in extreme environments can be studied if some black fungi were amenable to genetic manipulations. We selected the rock-inhabiting fungus Knufia petricola (class Eurotiomycetes, order Chaetothyriales) that grows moderately in axenic culture and exhibits all the characteristics of black yeasts such as yeast-like cell growth, absence of reproductive structures and constitutive dihydroxynaphthalene (DHN) melanogenesis. For this environmental strain we developed protocols to efficiently generate and transform protoplasts resulting in stable homokaryotic transformants by targeting genes involved in pigment synthesis and expressing fluorescent reporter genes. Hence, endogenous and foreign genes can be expressed from episomal AMA1-containing plasmids and genome-integrated DNA constructs. Moderate rates of homologous recombination allow for both ectopic and targeted integrations. CRISPR-Cas9 was further validated as a strategy for obtaining selection marker-free mutants and silencing via RNA interference as an approach to study essential genes. Availability of this genetic toolbox and an annotated genome sequence is paving the way for studying interactions of K. petricola and other black yeasts with environmental stressors, material surfaces, soil matrices and phototrophic symbionts.
Soiling of photovoltaic (PV) systems compromises their performance causing a significant power loss and demanding periodical cleaning actions. This phenomenon raises great concerns in the solar energy field, thus leading to notable research efforts over the last decades. Soiling is caused by a dual action of dust deposition and biofouling. However, surprisingly, the microbiological contribution to PV soiling is often overlooked or underestimated. In this study, a variety of qPCR-based methods have been developed to quantify the microbial load of fungi, bacteria and phototrophs on PV panels. These protocols were evaluated by comparison with culturedependent methods, and were implemented with real solar plants for two years. The results show that the developed molecular methods are highly sensitive and reliable to monitor the microbial component of the soiling. Fungal biomass was clearly dominant in all analysed PV modules, while bacteria and phototrophs showed much lower abundance. Light microscopy and qPCR results revealed that melanised microcolonial fungi and phototrophs are the main biofilm-forming microorganisms on the studied solar panels. In particular, the fungal qPCR protocol is proposed as a useful tool for monitoring of PV soiling, and investigating the microbial contribution to specific soiling cases.
Graphene and its derivatives have recently attracted much attention for sensing and deactivating pathogens. However, the mechanism of multivalent interactions at the graphene–pathogen interface is not fully understood. Since different physicochemical parameters of graphene play a role at this interface, control over graphene’s structure is necessary to study the mechanism of these interactions. In this work, different graphene derivatives and also zwitterionic graphene nanomaterials (ZGNMs) were synthesized with defined exposure, in terms of polymer coverage and functionality, and isoelectric points. Then, the switchable interactions of these nanomaterials with E. coli and Bacillus cereus were investigated to study the validity of the generally proposed “trapping” and “nano-knives” mechanisms for inactivating bacteria by graphene derivatives. It was found that the antibacterial activity of graphene derivatives strongly depends on the accessible area, i.e. edges and basal plane of sheets and tightness of their agglomerations. Our data clearly confirm the authenticity of “trapping” and “nano-knives” mechanisms for the antibacterial activity of graphene sheets.
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.
Microbial activity and functioning in soils are strongly limited by carbon (C) availability, of which a great proportion is released by living roots. Rhizodeposition and especially root exudates stimulate microbial activity and growth, and may shift the stoichiometric balance between C, N, and P. Thereby, exudates heighten microbial nutrient demand and acquisition of N and P from organic matter, leading to an increase in enzyme production. Aim of this study was to determine environmental controls of extracellular enzyme production, and hence on potential enzyme activities (Vmax) and substrate affinities (Km). To determine the controlling factors, we worked on four spatial scales from the microscale (i.e. rhizosphere) through the mesoscale (i.e. soil depth) and landscape scale (relief positions), and finally to the continental scale (1200 km transect within the Coastal Cordillera of Chile). Kinetics of seven hydrolyzing enzymes of the C, N, and P cycles (cellobiohydrolase, β‑glucosidase, β‑xylosidase, β‑N‑acetylglucosaminidase, leucine‑aminopeptidase, tyrosine‑aminopeptidase, and acid phosphatase) were related to soil texture, C and N contents, pH, and soil moisture via redundancy analysis (RDA). Potential activities of C, N, and P acquiring enzymes increased up to 7-times on the continental scale with rising humidity of sites and C and N contents, while substrate affinities simultaneously declined. On the landscape scale, neither Vmax nor Km of any enzyme differed between north and south slopes. From top- to subsoil (down to 120 cm depth) potential activities decreased (strongest of aminopeptidases under humid temperate conditions with up to 90%). Substrate affinities, however, increased with soil depth only for N and P acquiring enzymes. Affinities of cellobiohydrolase and β‑xylosidase, on the contrary, were 1.5- to 3-times higher in top- than in subsoil. Potential activities of N and P acquiring enzymes and β‑glucosidase increased form bulk to roots. Simultaneously, substrate affinities of N and P acquiring enzymes declined, whereas affinities of β‑glucosidase increased. These trends of activities and affinities in the rhizosphere were significant only for acid phosphatase. The RDA displayed a strong relation of potential activities of C and P acquiring enzymes and β‑N‑acetylglucosaminidase to C and N contents in soil as well as to the silt and clay contents. Aminopeptidase activity was mainly dependent on soil moisture and pH. We conclude that substrate availability for microorganisms mainly determined enzyme activity patterns on the continental scale by the humidity gradient. Patterns on the meso- and microscale are primarily controlled by nutrient limitation, which is induced by a shift of the stoichiometric balance due to input of easily available C by roots in the rhizosphere.
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.
The development of a test to evaluate the degradation of semi-volatile fuels as diesel by microorganisms is presented. This method is based on the principles described in the CEC-L-103 Standard procedure that is exclusively meant for testing the biodegradability of non-volatile lubricants. Therefore, significant modifications involve aseptic conditions for testing specific microorganisms and conducting the test in closed vessels avoiding evaporation losses, while fuel quantification using gas chromatography-flame ionization detection (GC-FID) is retained. It is suggested that the modified procedure should enable routine application for semi-volatile hydrocarbon-based fuels. GC-FID provides additionally valuable information on the alteration of fuel component patterns during biodegradation. The procedure was successfully tested using two bacteria (Pseudomonas aeruginosa and Sphingomonas sp.) and two yeasts (Moesziomyces sp. and Candida sp.) isolated from real diesel contamination cases. All tested microorganisms caused a significant degradation of diesel fuel achieving hydrocarbon degradation percentages ranging from 23% to 35%. Specific aspects on the test modification and prospects for further modification regarding targeted investigations in the field of fuel contamination by microorganisms are briefly discussed.
Active antibacterial and antifouling surface coating via a facile one-step enzymatic cross-linking
(2017)
Prevention of microbial contamination of surfaces is one of the biggest challenges for biomedical applications. Establishing a stable, easily produced, highly antibacterial surface coating offers an efficient solution but remains a technical difficulty. Here, we report on a new approach to create an in situ hydrogel film-coating on glass surfaces made by enzymatic cross-linking under physiological conditions. The cross-linking is catalyzed by horseradish peroxidase (HRP)/glucose oxidase (GOD)-coupled cascade reactions in the presence of glucose and results in 3D dendritic polyglycerol (dPG) scaffolds bound to the surface of glass.
These scaffolds continuously release H2O2 as long as glucose is present in the system. The resultant polymeric coating is highly stable, bacterial-repellent, and functions under physiological conditions. Challenged with high loads of bacteria (OD540 = 1.0), this novel hydrogel and glucose-amended coating reduced the cell viability of Pseudomonas putida (Gram-negative) by 100% and Staphylococcus aureus (Gram-positive) by ≥40%, respectively. Moreover, glucose-stimulated production of H2O2 by the coating system was sufficient to kill both test bacteria (at low titers) with >99.99% Efficiency within 24 h. In the presence of glucose, this platform produces a coating with high effectiveness against bacterial adhesion and survival that can be envisioned for the applications in the glucose-associated medical/oral devices.
Essential processes necessary for the establishment and maintenance of rock biofilms include photosynthesis, production of extracellular polymeric substances, substrate penetration and nutrient enrichment from the atmosphere. This natural functional diversity is supported by a complex biofilm community consisting of heterotrophic and phototrophic microorganisms. While heterotrophic rock biofilm-formers are dominated by stress-tolerant microcolonial ascomycetes, phototrophs might be represented by diverse algae and cyanobacteria. Reduction of model systems to the genetically tractable minimum, a proven successful experimental strategy in different symbiotic systems, is now applied to rock biofilms. These complex and important natural systems are simulated in the lab using genetic methods coupled with continuous microscopic and analytical (microscopic as well as geochemical and biochemical) observations. Our in vitro bipartite model includes free-living and symbiosis-competent, genetically tractable microorganisms – a rock-inhabiting fungus Knufia petricola A95 and a cyanobacterium Nostoc punctiforme strain ATCC 29133. To accurately reflect the development of a rock biofilm, contacts of these two genetically tractable partners are studied under well-controlled laboratory conditions. This experimental strategy is strongly supported by the knockout mutants of Knufia petricola which have been recently created and will be compared in their action on mineral surfaces. An impact of Knufia petricola strain A95 with or without protective pigments on mineral adhesion and alteration will be presented.
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.
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.
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.
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. Applied research is necessary to find ways of preventing MCF from colonising and degrading solar panels and historic monuments.
A wide variety of fungi and bacteria are known to contaminate fuels and fuel systems. These microbial contaminants have been linked to fuel system fouling and corrosion. The fungus Hormoconis resinae, a common jet fuel contaminant, is used in this study as a model for developing innovative risk assessment methods. A novel qPCR protocol to detect and quantify H. resinae in, and together with, total fungal contamination of fuel systems is reported. Two primer sets, targeting the markers RPB2 and ITS, were selected for their remarkable specificity and sensitivity. These primers were successfully applied on fungal cultures and diesel samples demonstrating the validity and reliability of the established qPCR protocol. This novel tool allows clarification of the current role of H. resinae in fuel contamination cases, as well as providing a technique to detect fungal outbreaks in fuel systems. This tool can be expanded to other well-known fuel-deteriorating microorganisms.
The interface between materials and the environment is populated by microorganisms which organize on surfaces to form specially adapted and resistant microbial associations, so-called biofilms. Surface-bound biofilm growth is associated with substantial secretion of metabolic products that can significantly influence material corrosion. Here, a group of material-inhabiting fungi are presented, which colonize and weather air-exposed materials such as building façades, roofs and solar systems. A selected model fungus is used to carry out genetic and molecular biology experiments, which are microbiologically cutting-edge and allow for the development of novel time-saving techniques for material testing. The degree of material damage under the influence of this model fungus shows a measure of the aggressiveness of the biofilm under defined environmental conditions - and thus explores fundamental components of the interaction between material and biofilm.
The interface between materials and the environment is populated by microorganisms which organize on surfaces to form specially adapted and resistant microbial associations, so-called biofilms. Surface-bound biofilm growth is associated with substantial secretion of metabolic products that can significantly influence material corrosion. Here, a group of material-inhabiting fungi are presented, which colonize and weather air-exposed materials such as building façades, roofs and solar systems. A selected model fungus is used to carry out genetic and molecular biology experiments, which are microbiologically cutting-edge and allow for the development of novel time-saving techniques for material testing. The degree of material damage under the influence of this model fungus shows a measure of the aggressiveness of the biofilm under defined environmental conditions - and thus explores fundamental components of the interaction between material and biofilm.
Genetic manipulation of protective pigments in a rock-inhabiting model fungus Knufia petricola A95
(2016)
Sub-aerial biofilms typically form on bare rock. They consist of 99% cell material and extracellular polymeric substances (EPS) metabolising at low water availability. Rock-inhabiting melanised fungi represent an important part of the microbial community in these environments, playing important roles in the colonisation of mineral surfaces, rock weathering and soil formation in the ecological/geochemical context. Different cellular stress responses make rock-inhabiting ascomycetes fit for survival under extremely changing irradiation, as well as water, energy sources and nutrient availability. Melanised, rock-inhabiting fungi possess multiple protective pigments, form facultative symbiotic associations with photobionts and weather minerals. Melanised fungi build a protective layer around the cell that is critical in adhesion to other living partners, for the colonisation of the substrate and in the subsequent damage of the colonised surface. We chose Knufia petricola (Chaetothyriales) as a model species to analyse colonisation of surfaces. The basic physiology of K. petricola strain A95 is studied, its full genome sequence has been prepared for annotation and methods for deleting specific genes have been established. Unique features of K. petricola including the protective pigments (melanin and carotenoids) and EPS/cell wall properties are now being dissected genetically. As K. petricola strain A95 is in the basic clade of Chaetothyriales, it is an ancestor of both important human pathogens including Exophiala and lichens from the Verrucariaceae family. For this reason studies with A95 can help clarify the basis of fungal pathogenicity – as well as explain interactions with microscopic phototrophic partners like unicellular green algae and cyanobacteria. With Knufia petricola we will establish a canon of experimental approaches to characterise and quantify fungi that actively contact inanimate solid materials. The set of methods developed for Knufia will be adapted to heavily melanised and EPS-producing ascomycetes and can be broadly applied to medically important as well as material-colonising fungi.
A new tool in material science - Targeted disruption of melanin synthesis in rock-inhabiting fungi
(2016)
The ability to survive almost absolute dehydration through air-drying is a remarkable feature of rock-inhabiting microcolonial fungi (MCF), which colonise rock surfaces in hot and cold deserts. Understanding of the underlying mechanisms which allow this group of fungi to conquer natural and man-made environments requires a set of modern biological techniques and approaches that are under development in our laboratory. We will present an overview of the targeted disruption of melanin biosynthesis genes in the rock-inhabiting Knufia petricola and give inside into the lines of research and the network of supporting laboratories that made this progress possible.
Fungal extracellular materials reinforce a constant interaction between their cell wall and the environment. A dynamic mixture of chitin, glucans, mannans, glycoproteins, glycolipids and pigments supports the success of all fungal life styles – from symbiotic to the free-living and pathogenic. Fungi are perfectly adapted to grow on surfaces and in porous environments, where they form medically and geochemically relevant biofilms. Fungal EPS are critical in adhesion to other fungi, other cells or substratum as well as in the following interaction with the host immune system or material they attack, degrade and deteriorate respectively. Characterisation of extracellular compounds and understanding of its function is necessary to limit damage caused by fungal activity. All necessary methodology from chemical characterization to complete genetic analyses has been developed for medically important fungi. Now it is time to apply this knowledge to the numerous, largely aerobic and very active organisms that occupy a wide range of atmosphere-exposed habitats in the upper lithosphere. One can expect that analogies between medically- and environmentally-relevant model fungal species will help us to address the dynamics of the fungal cell EPS matrix in much more efficient and widely applicable ways.
Essential processes in the establishment and maintenance of rock biofilms include photosynthesis, production of extracellular polymeric substances, substrate penetration and atmospheric nutrient enrichment. Functional diversity is supported by a complex subaerial biofilm (SAB) community of heterotrophic and phototrophic microorganisms. Stress tolerant and melanised Ascomycetes dominate heterotrophic SABs while diverse algae and cyanobacteria comprise the phototrophic consortia. Laboratory simulation of SABs permits use of molecular-genetic methods coupled with geochemical and microscopic analyses to study weathering. Our in vitro model includes two free-living and symbiosis-competent, genetically tractable microorganisms: the cyanobacterium Nostoc punctiforme and the microcolonial rock-inhabiting fungus Knufia petricola. This genetically amenable cyanobacterium/fungus model biofilm allows development of quantitative methods tailored to the natural diversity of SABs. Precise, reproducible studies using this model biofilm have shown that both the melanised fungus, as well as the combined bacterial/fungal system, enhances the weathering of minerals. Geochemical signatures of these in vitro rock biofilms can now be obtained and compared with bacterial/fungal mutants of varied EPS composition and substrate penetration patterns. And finally, precise study of the model cyanobacterium/fungus biofilm will Permit prediction of the effects of conservational treatments.
A biological impact on weathering was recognized already at the beginning of the twentieth century, when biochemical influence of the lichen growth on rocks was convincingly demonstrated. Later it was shown that the
progress of solid rock weathering initiated by biological colonization was affected by the initial porosity system and sensitivity of mineral association. In the meantime
a considerable amount of diverse scientific data confirm the importance of biological rock colonizers (lichens and free-living rock biofilms) in mineral material dynamics as they occur at the atmosphere-exposed rock surfaces on local as well as global scale. Subaerial rock biofilms—microbial ecosystem including free-living heterotrophic and phototrophic settlers of bare rock surfaces—are characteristic for the first stage of primary succession of terrestrial ecosystems on mineral substrates.
These cultivable and free-living communities are dominated by fungi and set the stage for the later development of a lichen cover, but in comparison to lichens also represent a new tool for laboratory experimentation and thus open a new stage of work in geomicrobiology. The Minerals sensitivity to microbially induced biological weathering can be demonstrated by studies of natural samples as well as by the laboratory mesocosm experiments.
Quantification of microbial load in diesel storage tanks using culture- and qPCR-based approaches
(2016)
Microbial contamination of fuels, associated with a wide variety of bacteria and fungi, leads to decreased product quality and can compromise equipment performance by biofouling and microbiologically influenced corrosion of pipelines and storage tanks. Detection and quantification of biomass are critical in monitoring fuel systems for an early detection of microbial outbreaks. The aims of this study are (i) to quantify bacterial and fungal contamination in samples from diesel storage tanks of petrol stations, using both culture dependent- and culture independent (qPCR) approaches, and (ii) to analyse the diversity of cultivable diesel-contaminating microorganisms with the purpose to create a strain collection for further use in biodeterioration experiments. Both methodological approaches revealed a high microbial contamination in all studied samples, with the bacterial load being much higher than the fungal load. The diversity of cultivable microorganisms was rather low. Based on criteria of abundance and fuel degradation potential, the most relevant microorganisms were identified as bacteria of genera Bacillus, Citrobacter, Burkholderia and Acetobacter, the filamentous fungi Paecilomyces variotii and Pseudallescheria boydii, and a Dipodascaceae yeast. Furthermore the validity and utility of qPCR-based methods are discussed.
Microbial colonization affects the efficiency of photovoltaic panels in a tropical environment
(2015)
Sub-aerial biofilm (SAB) development on solar panels was studied in São Paulo. After 6, 12 and 18 months' exposure, photovoltaic panels were covered by increasing proportions of organic matter (42%, 53% and 58%, respectively). Fungi were an important component of these biofilms; very few phototrophs were found. Major microorganisms detected were melanised meristematic ascomycetes and pigmented bacterial genera Arthrobacter and Tetracoccus. While diverse algae, cyanobacteria and bacteria were identified in biofilms at 6 and 12 months, diversity at a later stage was reduced to that typical for SAB: the only fungal group detected in 18 month biofilm was the meristematic Dothideomycetes and the only phototrophs Ulothrix and Chlorella. Photovoltaic modules showed significant power reductions after 6, 12 (both 7%) and 18 (11%) months. The lack of difference in power reduction between 6 and 12 months reflects the dual nature of soiling, which can result from the deposition of particulates as well as from SAB fouling. Although 12-month old SAB demonstrated an almost 10-fold increase in fungal colonization and a higher organic content, the larger non-microbial particles (above 10 µm), which were important for efficiency reduction of lightly-biofilmed panels, were removed by high rainfall just before the 12-month sampling.
Sub-aerial biofilms (SABs) are ubiquitous microbial communities that develop at the interface between hard surfaces and the atmosphere. Inherent SAB 'core-settlers' include phototrophic algae, cyanobacteria, heterotrophic bacteria and microcolonial fungi (MCF). SABs do not simply cover hard surfaces; they interact with them in myriads of ways and bind to the underlying substrate. Secretion of extracellular mucilage aids adhesion, while organic acids and acidic polysaccharides weather the surface. As protection against solar radiation, many members of the SAB consortia produce shielding pigments while the phototrophic inhabitants are laden with photosynthetic pigments. All absorb light of many wavelengths and in addition, the cells themselves scatter light. Both effects change the spectra of incoming radiation (including wavelengths that are converted to electricity by photovoltaic cells) and decrease its intensity. To quantify these effects on SABs as complex entities of organisms and pigments, we measured the spectral properties of model and natural biofilms transferred to glass. Here we show that SABs growing on solar panels and other substrates scatter incident radiation between 250 nm up to 1800 nm and block up to 70% of its transmission. Model biofilms have the advantage that their microbial components can be 'tuned' to resemble natural ones of different compositions thus providing a novel materials-testing tool.
Ancient mariners knew that dust whipped up from deserts by strong winds travelled long distances, including over oceans. Satellite remote sensing revealed major dust sources across the Sahara. Indeed, the Bodélé Depression in the Republic of Chad has been called the dustiest place on earth. We analysed desert sand from various locations in Chad and dust that had blown to the Cape Verde Islands. High throughput sequencing techniques combined with classical microbiological methods showed that the samples contained a large variety of microbes well adapted to the harsh desert conditions. The most abundant bacterial groupings in four different phyla included: (a) Firmicutes—Bacillaceae, (b) Actinobacteria—Geodermatophilaceae, Nocardiodaceae and Solirubrobacteraceae, (c) Proteobacteria—Oxalobacteraceae, Rhizobiales and Sphingomonadaceae, and (d) Bacteroidetes—Cytophagaceae. Ascomycota was the overwhelmingly dominant fungal group followed by Basidiomycota and traces of Chytridiomycota, Microsporidia and Glomeromycota. Two freshwater algae (Trebouxiophyceae) were isolated. Most predominant taxa are widely distributed land inhabitants that are common in soil and on the surfaces of plants. Examples include Bradyrhizobium spp. that nodulate and fix nitrogen in Acacia species, the predominant trees of the Sahara as well as Herbaspirillum (Oxalobacteraceae), a group of chemoorganotrophic free-living soil inhabitants that fix nitrogen in association with Gramineae roots. Few pathogenic strains were found, suggesting that African dust is not a large threat to public health.
The influence of material properties on bacterial attachment to surfaces needs to be understood when applying polymer-based biomaterials. Positively charged materials can kill adhered bacteria when the charge density is sufficiently high but such materials initially increase the adherence of some bacteria such as Escherichia coli. On the other hand, negatively charged materials have been shown to inhibit initial bacterial adhesion, but this effect has only been demonstrated in relatively few biomaterial classes and needs to be evaluated using additional systems. Gradients in surface charge can impact bacterial adhesion and this was tested in our experimental setup.
Moreover, the evaluation of bacterial adhesion to biomaterials is required to assess their potential for biological applications. Here, we studied the bacterial adhesion of E. coli and Bacillus subtilis on the surfaces of acrylonitrile-based copolymer samples with different amounts of 2-methyl-2-propene-1-sulfonic acid sodium salt (NaMAS) comonomer. The content related to NaMAS based repeating units nNaMAS varied in the range from 0.9 to 1.5 mol%.
We found a reduced colonized area of E. coli for NaMAS containing copolymers in comparison to pure PAN materials, whereby the bacterial colonization was similar for copolymers with different nNaMAS amounts. A different adhesion behavior was obtained for the second tested organism B. subtilis, where the implementation of negative charges into PAN did not change the overall adhesion pattern. Furthermore, it was observed that B. subtilis adhesion was significantly increased on copolymer samples that exhibited a more irregular surface roughness.
Rock-inhabiting black fungi [also microcolonial or meristematic fungi (MCF)] are a phylogenetically diverse group of melanised ascomycetes with distinctive morphological features that confer extensive stress tolerance and permit survival in hostile environments. The MCF strain A95 Knufia petricola (syn. Sarcinomyces petricola) belongs to an ancestral lineage of the order Chaetothyriales (class Eurotiomycetes). K. petricola strain A95 is a rock-inhabiting MCF and its growth requirements were studied using the 96-well plate-based Biolog System under ~1070 different conditions (osmotic stress, pH growth optima, growth factor requirements and nutrient catabolism). A95 is an osmotolerant, oligotrophic MCF that grows best around pH 5. Remarkably, A95 shows metabolic activity in the absence of added nitrogen, phosphorus or sulphur. Correlations could be drawn between the known nutrient requirements of A95 and what probably is available in sub-aerial systems (rock and other material surfaces). Detailed knowledge of A95's metabolic requirements allowed formulation of a synthetic medium that supports strong fungal growth.
Worldwide, abandoned coal mines release substantial amounts of methane, which is largely of biogenic origin. The aim of this study was to understand the microbial processes involved in mine-gas formation. Therefore, coal and timber samples and anaerobic enrichments from two abandoned coal mines in Germany were subjected to DGGE analyses and quantitative PCR. The primers used were specific for Bacteria, Archaea, Fungi, and the key functional genes for sulfate reduction (dsrA) and methanogenesis (mcrA). A broad spectrum of facultative anaerobic bacteria and acetogens belonging to all five groups (α-ε) of the Proteobacteria, as well as the Bacteroidetes, Tenericutes, Actinobacteria, Chlorobi and Chloroflexi were detected. Archaea were represented by acetoclastic Methanosarcinales and Crenarchaeota with an unknown metabolism. Fungi formed thick biofilms particularly on timber, and were identified as typical wood degraders belonging to the Ascomycetes and Basidiomycetes. The community analysis as well as the environmental conditions and the metabolites detected in a previous study are consistent with the following scenario of methane release: Weathering of coal and timber is initiated by wood-degrading Fungi and Bacteria under a suboxic atmosphere. In the lower, oxygen-depleted layers Fungi and Bacteria perform incomplete oxidation and release reduced substrates which can be channeled into methanogenesis. Acetate appeared to be the main precursor of the biogenic methane in the investigated coal mines.
Three principally different mechanisms contribute to the wear-down process of mineral aggregates in sedimentary environments: (1) mechanical abrasion by forces of wind and water and by floating or saltating neighbouring grains, (2) chemical attack and dissolution by fluids, and (3) physical bioerosion and chemical biocorrosion. It is however, difficult to attribute the specific surface changes to specific environments and processes. Quartz sand grains from subaerial and subaquatic environments were analysed by atomic force microscopy (AFM) for traces of natural and experimental aeolian, aquatic and biological wear-down processes. Quantitative topographical parameters of surface alterations were extracted from topography data by non-linear methods derived from digital image analysis. These parameters were examined by multivariate statistic, yielding three well-distinguishable groups. Morphological surface alterations dominated by subaerial, subaquatic and by biological impact could be differentiated. The method may also be used for the detection of aeolian, subaquatic, and biological modification of sedimentary grains and rock surfaces in extraterrestrial environments, and for assessment of environmental damage on monuments and buildings.
Biogenic impact on materials
(2011)
Materials as constituents of products or components of technical systems rarely exist in isolation and many must cope with exposure in the natural world. This chapter describes methods that simulate how a material is influenced through contact with living systems such as microorganisms and arthropods. Both unwanted and desirable interactions are considered. This biogenic impact on materials is intimately associated with the environment to which the material is exposed (Materials-Environment Interaction, Chap. 15). Factors such as moisture, temperature and availability of food sources all have a significant influence on biological systems. Corrosion (Chap. 12) and wear (Chap. 13) can also be induced or enhanced in the presence of microorganisms. Section 14.1 introduces the categories between desired (biodegradation) and undesired (biodeterioration) biological effects on materials. It also introduces the role of biocides for the protection of materials. Section 14.2 describes the testing of wood as a building material especially against microorganisms and insects. Section 14.3 characterizes the test methodologies for two other groups of organic materials, namely polymers (Sect. 14.3.1) and paper and textiles (Sect. 14.3.2). Section 14.4 deals with the susceptibility of inorganic materials such as metals (Sect. 14.4.1), concrete (Sect. 14.4.2) and ceramics (Sect. 14.4.3) to biogenic impact. Section 14.5 treats the testing methodology concerned with the performance of coatings and coating materials. In many of these tests specific strains of organisms are employed. It is vital that these strains retain their ability to utilize/attack the substrate from which they were isolated, even when kept for many years in the laboratory. Section 14.6 therefore considers the importance of maintaining robust and representative test organisms that are as capable of utilizing a substrate as their counterparts in nature such that realistic predictions of performance can be made.