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Eingeladener Vortrag
- nein (51)
Life at the atmosphere-lithosphere boundary is an ancient terrestrial niche that is sparsely covered by thin subaerial biofilms. The microbial inhabitants of these biofilms (a) have adapted to all types of terrestrial/subaerial stresses (e.g., desiccation, extreme temperatures, low nutrient availability, intense solar radiation), (b) interact with minerals that serve as both a dwelling and a source of mineral nutrients, and (c) provoke weathering of rocks and soil formation. Subaerial communities comprise heterotrophic and phototrophic microorganisms that support each other's lifestyle. Major lineages of eubacteria associated with the early colonization of land (e.g., Actinobacteria, Cyanobacteria) are present in these habitats along with eukaryotes such as microscopic green algae and ascomycetous fungi. The subaerial biofilm inhabitants have adapted to desiccation, solar radiation, and other environmental challenges by developing protective, melanized cell walls, assuming microcolonial architectures and symbiotic lifestyles. How these changes occurred, their significance in soil formation, and their potential as markers of climate change are discussed below.
The class Dothideomycetes (along with Eurotiomycetes) includes numerous rock-inhabiting fungi (RIF), a group of ascomycetes that tolerates surprisingly well harsh conditions prevailing on rock surfaces. Despite their convergent morphology and physiology, RIF are phylogenetically highly diverse in Dothideomycetes. However, the positions of main groups of RIF in this class remain unclear due to the lack of a strong phylogenetic framework. Moreover, connections between rock-dwelling habit and other lifestyles found in Dothideomycetes such as plant pathogens, saprobes and lichen-forming fungi are still unexplored. Based on multigene phylogenetic analyses, we report that RIF belong to Capnodiales (particularly to the family Teratosphaeriaceae s.l.), Dothideales, Pleosporales, and Myriangiales, as well as some uncharacterised groups with affinities to Dothideomycetes. Moreover, one lineage consisting exclusively of RIF proved to be closely related to Arthoniomycetes, the sister class of Dothideomycetes. The broad phylogenetic amplitude of RIF in Dothideomycetes suggests that total species richness in this class remains underestimated. Composition of some RIF-rich lineages suggests that rock surfaces are reservoirs for plant-associated fungi or saprobes, although other data also agree with rocks as a primary substrate for ancient fungal lineages. According to the current sampling, long distance dispersal seems to be common for RIF. Dothideomycetes lineages comprising lichens also include RIF, suggesting a possible link between rock-dwelling habit and lichenisation.
Solar energy has been transformed into useful redox differences or disequilibria within the Earth´s crust since the onset of anoxygenic and oxygenic photosynthesis in the Precambrian. Inorganic oxidized carbon is transformed into reduced carbon compounds by capturing and storing solar energy. During this process, many different organic compounds are formed including carbohydrates, proteins, hydrocarbons, and various other complex organic metabolic products and their diagenetic polymerization products (melanin, humic substances, petroleum, coal, and kerogen). Many of these solar energy-enriched compounds, however, are oxidized immediately or during the diagenetic transformation of sediments. The oxidation agents are oxygen, sulfate, iron, and other oxidized compounds, which in turn are partly enriched with the original solar energy. On a global biogeochemical scale, however, sulfur and iron are the most important elements. Geological evidence shows that biogeochemical cycles tend to yield stable ratios between the most oxidized forms of carbon (carbon dioxide and calcium/magnesium carbonate) and the most reduced forms (diamond, coal, methane, and hydrocarbons). Throughout the Earths history and evolution, this equilibrium ratio is around 1:4, maximally 1:5. When too much carbon is stored in the crust in the form of reduced compounds or vice versa, climatic and biogeomorphogenetic consequences upset the equilibrium. The biosphere reacts in a way to return to the optimal ratio. Excellent examples for this fluctuating equilibrium are the Carboniferous (too much organic carbon stored), the Permian (too little organic carbon stored), and the Tertiary with a generally equivalent production of hydrocarbons and carbonates. At present, we are in a period in which there is a global biogeochemical need to oxidize reduced carbon compounds as fast as possible in order to avoid even more dramatic global climate shifts. The highly evolved human genome seems to be the tool for this shift. Enormous amounts of reduced carbon are turned into the oxidized form as carbon dioxide, which by various biogeochemical pathways is quickly transformed into carbonate, another oxidized form of carbon that can be stabilized and stored in the sedimentary record. Fast recycling of excessively stored solar energy may enable the survival of a global biosphere under highly stressed conditions.
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.
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.
A laboratory biofilm consisting of the phototrophic cyanobacterium Nostoc punctiforme ATCC 29133 and the rock-inhabiting ascomycete Knufia petricola CBS 726.95 was tested for its mineral weathering potential. Minerals with different grain sizes and mineralogy were incubated with and without biofilm in batch and in flow-through column experiments. After incubation, the mineral dissolution was quantified analysing (i) leachate chemistry via ICP-OES/MS (inductively coupled plasma optical emission spectrometry/mass spectrometry) and (ii) the residual grains as thin polished sections via SEM/TEM-EDX (scanning electron microscopy/transmission electron microscopy-energy dispersive X-ray spectrometry). Mineral dissolution was enhanced in biotic experiments as compared to abiotic ones, for both batch culture and flow-through approaches. Analyses of thin polished sections confirmed the leaching of these elements near the surface of the mineral grains. These results clearly indicate a biotic effect on the weathering of minerals produced by the laboratory biofilm.
Polyglycerol (PG) coated polypropylene (PP) films were synthesized in a two-step approach that involved plasma bromination and subsequently grafting hyperbranched polyglycerols with very few amino functionalities. The influence of different molecular weights and density of reactive linkers were investigated for the grafted PGs. Longer bromination times and higher amounts of linkers on the surface afforded long-term stability. The protein adsorption and bacteria attachment of the PP-PG films were studied. Their extremely low amine content proved to be beneficial for preventing bacteria attachment.
A novel Gram-positive, aerobic, actinobacterial strain, CF5/5, was isolated from soil in the Sahara desert, Chad. It grew best at 20–35 °C and at pH 6.0–8.0 and with 0–4 % (w/v) NaCl, forming black-colored colonies. Chemotaxonomic and molecular characteristics of the isolate matched those described for members of the genus Geodermatophilus. The DNA G + C content was 75.9 mol%. The peptidoglycan contained meso-diaminopimelic acid; galactose and xylose were detected as diagnostic sugars. The main phospholipids were diphosphatidylglycerol, phosphatidylcholine, and phosphatidylinositol; MK-9(H4) was the dominant menaquinone. The major cellular fatty acids were: iso-C16:0 and iso-C15:0. The 16S rRNA gene showed 95.6–98.3 % sequence similarity with the other named members of the genus Geodermatophilus. Based on the polyphasic taxonomy data, the isolate is proposed to represent a novel species, Geodermatophilus saharensis with the type strain CF5/5T = DSM 45423 = CCUG 62813 = MTCC 11416.
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.
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 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.
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.
Biofilm formation on materials leads to high costs in industrial processes, as well as in medical applications. This fact has stimulated interest in the development of new materials with improved surfaces to reduce bacterial colonization. Standardized tests relying on statistical evidence are indispensable to evaluate the quality and safety of these new materials. We describe here a flow chamber system for biofilm cultivation under controlled conditions with a total capacity for testing up to 32 samples in parallel. In order to quantify the surface colonization, bacterial cells were DAPI (4‘,6-diamidino-2-phenylindole)-stained and examined with epifluorescence microscopy. More than 100 images of each sample were automatically taken and the surface coverage was estimated using the free open source software g’mic, followed by a precise statistical evaluation. Overview images of all gathered pictures were generated to dissect the colonization characteristics of the selected model organism Escherichia coli W3310 on different materials (glass and implant steel). With our approach, differences in bacterial colonization on different materials can be quantified in a statistically validated manner. This reliable test procedure will support the design of improved materials for medical, industrial, and environmental (subaquatic or subaerial) applications.
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.
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.
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.
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 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.
Melanised cell walls and extracellular polymeric matrices protect rock-inhabiting microcolonial fungi from hostile environmental conditions. How extracellular polymeric substances (EPS) perform this protective role was investigated by following development of the model microcolonial black fungus Knufia petricola A95 grown as a sub-aerial biofilm. Extracellular substances were extracted with NaOH/formaldehyde and the structures of two excreted polymers studied by methylation as well as NMR analyses. The main polysaccharide (~ 80%) was pullulan, also known as α-1,4-; α-1,6-glucan, with different degrees of polymerisation. Αlpha-(1,4)-linked-Glcp and α-(1,6)-linked-Glcp were present in the molar ratios of 2:1. A branched galactofuromannan with an α-(1,2)-linked Manp main chain and a β-(1,6)-linked Galf side chain formed a minor fraction (~ 20%). To further understand the roles of EPS in the weathering of minerals and rocks, viscosity along with corrosive properties were studied using atomic force microscopy (AFM). The kinetic viscosity of extracellular K. petricola A95 polysaccharides (≈ 0.97 × 10-6 m2 s-1) ranged from the equivalent of 2% (w/v) to 5% glycerine, and could thus profoundly affect diffusion-dominated processes. The corrosive nature of rock-inhabiting fungal EPS was also demonstrated by its effects on the aluminium coating of the AFM cantilever and the silicon layer below.
A novel Gram-positive, aerobic, actinobacterial strain, CF6/1T, was isolated in 2007 during environmental screening of arid desert soil in the Sahara near to Ourba, Chad. The isolate was found to grow best in a temperature range of 20–37 °C and at pH 6.0–8.5 and showed no NaCl tolerance, forming black-coloured and nearly circular colonies on GYM agar. Chemotaxonomic and molecular characteristics determined for the isolate match those previously described for members of the genus Geodermatophilus. The DNA G + C content of the novel strain was determined to be 74.9 mol %. The peptidoglycan was found to contain meso-diaminopimelic acid as the diagnostic diamino acid. The main phospholipids were determined to be phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine, diphosphatidylglycerol and traces of phosphatidylglycerol; MK-9(H4) was identified as the dominant menaquinone and galactose as the diagnostic sugar. The major cellular fatty acids were found to be the branched-chain saturated acids iso-C16:0 and iso-C15:0, as well as C17:1ω8c. The 16S rRNA gene sequence shows 97.5–97.9 % sequence identity with the four validly named or at least effectively published members of the genus: Geodermatophilus obscurus (97.5 %), Geodermatophilus arenarius (97.7 %), Geodermatophilus ruber (97.9 %) and Geodermatophilus nigrescens (97.9 %). Based on the results from this polyphasic taxonomic analysis and DNA–DNA hybridizations with all type strains of the genus, we propose that strain CF6/1T represents a novel species, Geodermatophilus siccatus, with the type strain CF6/1T = DSM 45419T = CCUG 62765T = MTCC 11414T.
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.
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.
Geodermatophilus africanus sp. nov., a halotolerant actinomycete isolated from Saharan desert sand
(2013)
A novel Gram-strain positive, aerobic, actinobacterial strain, designated CF11/1T, was isolated from a sand sample obtained in the Sahara Desert, Chad. The black-pigmented isolate was aerobic and exhibited optimal growth from 25 to 35 °C at pH 6.08.0 and with 08 % (w/v) NaCl, indicating that it is a halotolerant mesophile. Chemotaxonomic and molecular characteristics of the isolate matched those described for members of the genus Geodermatophilus. The G+C content in the genome was 74.4 mol%. The peptidoglycan contained meso-diaminopimelic acid as diagnostic diaminoacid. The main phospholipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and a minor fraction of phosphatidylglycerol; MK-9(H4) was the dominant menaquinone, and galactose was detected as a diagnostic sugar. The major cellular fatty acid was branched-chain saturated acid iso-C16:0. Analysis of 16S rRNA gene sequences showed 95.398.6 % pairwise sequence identity with the members of the genus Geodermatophilus. Based on phenotypic and chemotaxonomic properties, as well as phylogenetic distinctiveness, the isolate represents a novel species, Geodermatophilus africanus, with the type strain CF11/1T (DSM 45422 = CCUG 62969 = MTCC 11556).
A novel Gram-reaction-positive actinobacterial strain, designated CF5/3T, was isolated from a sand sample obtained in the Sahara Desert, Chad. The greenish-black-pigmented isolate was aerobic and exhibited optimal growth from 25–40 °C at pH 6.0–10.0 with 0–1 % (w/v) NaCl. Chemotaxonomic and molecular characteristics of the isolate matched those described for members of the genus Geodermatophilus. The DNA G+C content of the genome of the novel strain was 75.5 mol%. The peptidoglycan contained meso-diaminopimelic acid as diagnostic diamino acid. The main phospholipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and a minor fraction of phosphatidylglycerol. MK-9(H4) was the dominant menaquinone, and galactose was detected as a diagnostic sugar. The major cellular fatty acids were branched-chain saturated acids: iso-C15:0 and iso-C16:0. Analysis of 16S rRNA gene sequences showed 95.6–98.8 % pairwise sequence identity with the members of the genus Geodermatophilus. Based on phenotypic and chemotaxonomic properties, as well as phylogenetic distinctiveness, the isolate represents a novel species, Geodermatophilus normandii, with the type strain CF5/3T (=DSM 45417T=CCUG 62814T=MTCC 11412T).
A novel Gram-positive, multiloculated thalli-forming, aerobic, actinobacterial strain, CF9/1/1T, was isolated in 2007 during environmental screening for xerophilic fungi in arid desert soil from the Sahara desert, Chad. The isolate grew best at a temperature range of 20–35 °C and at pH 6.0–8.5 and with 0–4% (w/v) NaCl, forming black-coloured and irregular colonies on GYM agar. Chemotaxonomic and molecular characteristics of the isolate matched those described for members of the genus Geodermatophilus. The DNA G+C content of the novel strain was 75.4 mol%. The peptidoglycan contained meso-diaminopimelic acid as a diagnostic diamino acid. The main phospholipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, a not yet structurally identified aminophospholipid and a small amount of phosphatidylglycerol; MK-9(H4) was identified as the dominant menaquinone and galactose was a diagnostic sugar. The major cellular fatty acids were branched-chain saturated acids: iso-C16:0 and iso-C15:0. The 16S rRNA gene sequence of the isolate showed 94.697.0% sequence similarities with those of five members of the genus: Geodermatophilus ruber DSM 45317T (94.6%), Geodermatophilus obscurus DSM 43160T (94.8%), Geodermatophilus siccatus DSM 45419T (96.2%), Geodermatophilus nigrescens DSM 45408T (96.7%) and Geodermatophilus arenarius DSM 45418T (97.0%). Based on the evidence from this polyphasic taxonomic study, a novel species, Geodermatophilus telluris sp. nov., is proposed; the type strain is CF9/1/1T (=DSM 45421T=CCUG 62764T).
Luftstaub über den Meeren - Wie historische Staubproben Wissenschaftler und Künstler inspirieren
(2013)
Subaerial biofilms (SAB) are an important factor in weathering, biofouling, and biodeterioration of bare rocks, building materials, and solar panel surfaces. The realm of SAB is continually widened by modern materials, and the settlers on these exposed solid surfaces always include melanized, stress-tolerant microcolonial ascomycetes. After their first discovery on desert rock surfaces, these melanized chaetothyrialean and dothidealean ascomycetes have been found on Mediterranean monuments after biocidal treatments, Antarctic rocks and solar panels. New man-made modifications of surfaces (e.g., treatment with biocides or photocatalytically active layers) accommodate the exceptional stress-tolerance of microcolonial fungi and thus further select for this well-protected ecological group. Melanized fungal strains were isolated from a microbial community that developed on highly photocatalytic roof tiles after a long-term environmental exposure in a maritime-influenced region in northwestern Germany. Four of the isolated strains are described here as a novel species, Constantinomyces oldenburgensis, based on multilocus ITS, LSU, RPB2 gene phylogeny. Their closest relative is a still-unnamed rock-inhabiting strain TRN431, here described as C. patonensis. Both species cluster in Capnodiales, among typical melanized microcolonial rock fungi from different stress habitats, including Antarctica. These novel strains flourish in hostile conditions of highly oxidizing material surfaces, and shall be used in reference procedures in material testing.
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.
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.
Magura Cave, north-western Bulgaria, possesses valuable rock-art paintings made with bat guano and dated from the period between the Eneolithic and Bronze Ages. Since 2008, the Art Gallery is closed to the general public in order to protect the paintings from vandalism, microclimatic changes caused by visitors and artificial illumination, and the consequent growth of fungi and phototrophs. Nevertheless, some tourist visits are allowed under the supervision of cave managers. This study provides the first scientific report on cultivable fungal assemblages dwelling different substrata in the Art Gallery. A total of 78 strains, belonging to 37 OTUs (Ascomycota 81%, Zygomycota 13%, Basidiomycota 5%), were isolated in the study. This fungal diversity was clearly dominated by Penicillium (50% of strains) and Aspergillus (13%). The most relevant visible fungal colonies were detected in sediments rich in bat guano, where, besides Penicillium, other guanophilic fungi such as Mucor, Mortierella, Trichosporon and Trichoderma were dominant. Conversely, scarce fungi were detected on rock surface of painted walls. Based on the biocide susceptibility assay, octylisothiazolinone (OIT) and benzalkonium chloride (BAC) were effective inhibiting the in vitro growth of dominant fungal species in Magura Cave, when applied at concentrations ranged from 100 to 1,000 mg/L. These data provide a valuable knowledge about Magura fungi, and exemplify a type of preliminary test that may be conducted before planning any biocide treatment. However, considering the irreversible effects of biocides on the ecological balance in caves, and the low fungal contamination in painted walls of Magura Cave, there is no reason to use conventional biocides in this cave. Further studies, monitoring microbial communities and microclimatic parameters, should be conducted to improve the knowledge on microbial ecology in Magura Cave and possible human impacts, as well as to allow the early detection of potential microbial outbreaks.
Genetic transformation of Knufia petricola A95 - a model organism for biofilm-material interactions
(2014)
We established a protoplast-based system to transfer DNA to Knufia petricola strain A95, a melanised rock-inhabiting microcolonial fungus that is also a component of a model sub-aerial biofilm (SAB) system. To test whether the desiccation resistant, highly melanised cell walls would hinder protoplast formation, we treated a melanin-minus mutant of A95 as well as the type-strain with a variety of cell-degrading enzymes. Of the different enzymes tested, lysing enzymes from Trichoderma harzianum were most effective in producing protoplasts. This mixture was equally effective on the melanin-minus mutant and the type-strain. Protoplasts produced using lysing enzymes were mixed with polyethyleneglycol (PEG) and plasmid pCB1004 which contains the hygromycin B (HmB) phosphotransferase (hph) gene under the control of the Aspergillus nidulans trpC. Integration and expression of hph into the A95 genome conferred hygromycin resistance upon the transformants. Two weeks after plating out on selective agar containing HmB, the protoplasts developed cell-walls and formed colonies. Transformation frequencies were in the range 36 to 87 transformants per 10 µg of vector DNA and 106 protoplasts. Stability of transformation was confirmed by sub-culturing the putative transformants on selective agar containing HmB as well as by PCR-detection of the hph gene in the colonies. The hph gene was stably integrated as shown by five subsequent passages with and without selection pressure.
Introduction. Throughout a remarkably long period land colonisation on Earth has been proceeding by biofilm growth on bare rock surfaces. This very long history of sub-aerial biofilm development resulted in a high degree of their specialisation in challenging environments including desert rocks and high mountain altitudes. Presently the solid substrate/atmosphere interface habitat is frequently anthropogenic and includes e.g. building surfaces as well as industrial energy-producing facilities like solar plants. Those can be looked upon as pseudodeserts inhabited by complex microbial communities metabolising under limited water availability and high sun irradiation.
Aim. We intended to compare microbial settlers from sub-aerial biofilms that grow on photovoltaic panels and painted building facades surfaces to the known database of the rock-inhabiting fungi.
Materials and methods. Characteristic organisms' that dominate specific rock-inhabiting as well as buildings' facades- and photovoltaic panel-dwelling communities were isolated, identified, and characterized by microbiological and molecular biological methods.
Results. Melanised meristematic ascomycetes are the most enduring and numerous dwellers on sub-aerial rock, facade and solar panel surfaces. Obviously, environmental changes perturb sub-aerial biofilm development but over a number of seasons, these changes result in relatively stable microbial communities peculiar to this particular environmental niche.
Conclusions. A broad selection of melanised meristematic ascomycetes is indicative of sub-aerial biofilm on all atmosphere-exposed surfaces and can thus be referred to as "reference organisms" for these habitats. Our experimental evidence confirms that colonization of such substrates is facilitated by a symbiosis between a photosynthesizing organism and a fungus that are equipped to cope with the stress associated with sub-aerial existence. Melanised ascomycetes possess a very special stress-tolerant life style that arises under the influence of atmosphere and the solid support. It should be pointed out, that (1) these biofilm communities cannot be considered as "primitive" ones regarding the very long history of their development and high degree of their specialization; and (2) symbiotically competent but free-living bacterial/fungal biofilms cannot be compared to lichen communities, evolving much later than biofilm ecosystems. A genetically tractable laboratory system that includes the key participants of sub-aerial biofilm ecosystems is currently used for the development of standard test procedures in material sciences.
Keywords. Sub-aerial biofilms, rock-inhabiting melanised fungi, reference organisms, solar panels biofilms
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.
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.
Desert dust seeds distant lands and waters
with minerals as well as micro-organisms raising the
question of whether this ancient phenomenon also
spreads pathogens across the globe. Severe dust
storms require strong winds blowing over land-masses
that are largely devoid of vegetation, effectively
limiting the scope for winds to raise pathogens into
the air. Nevertheless, changing patterns of land-use,
often driven by belligerency, result in refugees
spreading to areas that were previously deemed barely
habitable. With the help of the International Committee
of the Red Cross, a number of sand/dust samples
were collected from the Republic of Chad, some near
refugee camps, others further removed from human
influence. In parallel studies, we documented the
micro-organisms present in these samples and used a
number of the isolates here to test the effect of
environmental constraints on their ability to survive
intercontinental flight. We also added traditional
pathogens to the palette of microbes and tested the
effects of UV irradiation, desiccation and temperature
on survival of both bacteria and fungi. A clear trend
was obvious—those microbes that are coloured or able
to form conidia or spores (in other words, those that
are native to deserts) were well able to resist the
imposed stresses. On the other hand, most pathogens
were more sensitive to stresses than the environmental
isolates. Toxin production in two species of Aspergillus
was also investigated. Short-term desiccation
(simulating environmental conditions during intercontinental
travel) of sand amended with fungal spores
containing sterigmatocystin leads to increased mycotoxin
contents, but significant mycotoxin production
was only possible under growth-permissive conditions,
e.g. at higher humidity. It thus seems likely that an ever-decreasing fraction of the initial pathogen load
survives as the dust recedes from its desert source and
that those organisms that land on other continents are
highly enriched in desert dwellers.
A novel Gram-positive, aerobic, actinobacterial strain, CF5/4T, was isolated in 2007 during an environmental screening of arid desert soil in Ouré Cassoni, Chad. The isolate grew best in a temperature range of 2840 °C and at pH 6.0-8.5, with 0-1 % (w/v) NaCl, forming brown-coloured and nearly circular colonies on GYM agar. Chemotaxonomic and molecular characteristics of the isolate matched those described for members of the genus Geodermatophilus. The DNA G + C content of the novel strain was 75.9 mol %. The peptidoglycan contained meso-diaminopimelic acid as diagnostic diaminoacid. The main phospholipids were phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, diphosphatidylglycerol and a small amount of phosphatidylglycerol; MK-9(H4) was identified as the dominant menaquinone and galactose as diagnostic sugar. The major cellular fatty acids were branched-chain saturated acids: iso-C15:0 and iso-C16:0. The 16S rRNA gene showed 96.298.3 % sequence identity with the three members of the genus Geodermatophilus: G. obscurus (96.2 %), G. ruber (96.5 %), and G. nigrescens (98.3 %). Based on the chemotaxonomic results, 16S rRNA gene sequence analysis and DNA–DNA hybridization with the type strain of G. nigrescens, the isolate is proposed to represent a novel species, Geodermatophilus arenarius (type strain CF5/4T = DSM 45418T = MTCC 11413T = CCUG 62763T).
Microbiological studies on the intercontinental transport of dust are confounded by the difficulty of obtaining sufficient material for analysis. Axenic samples of dust collected at high altitudes or historic specimens in museums are often so small and precious that the material can only be sacrificed when positive results are assured. With this in mind, we evaluated current methods and developed new ones in an attempt to catalogue all microbes present in small dust or sand samples. The methods used included classical microbiological approaches in which sand extracts were plated out on a variety of different media, polymerase chain reaction (PCR)-based amplification of 16S/18S rRNA sequences followed by construction of clone libraries, PCR amplification of 16S rRNA sequences followed by high-throughput sequencing (HtS) of the products and direct HtS of DNA extracted from the sand. A representative sand sample collected at Bahaï Wadi in the desert of the Republic of Chad was used. HtS with or without amplification showed the most promise and can be performed on ≤100 ng DNA. Since living microbes are often required, current best practices would involve geochemical and microscopic characterisation of the sample, followed by DNA isolation and direct HtS. Once the microbial content of the sample has been deciphered, growth conditions (including media) can be tailored to isolate the micro-organisms of interest.
Three novel Gram-positive, aerobic, actinobacterial strains, CF5/2T, CF5/1 and CF7/1, were isolated in 2007 during environmental screening of arid desert soil in the Sahara desert, Chad. Results from riboprinting, MALDI-TOF protein spectra and 16S rRNA sequence analysis confirmed that all three strains belonged to the same species. Phylogenetic analysis of 16S rRNA sequences with the strains' closest relatives indicated that they represented a distinct species. The three novel strains also shared a number of physiological and biochemical characteristics distinct from previously named Geodermatophilus species. The novel strains' peptidoglycan contained meso-diaminopimelic acid; their main phospholipids were phosphatidylcholine, phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol and a small amount of phosphatidylglycerol; MK-9(H4) was the dominant menaquinone. The major cellular fatty acids were the branched-chain saturated acids iso-C16:0 and iso-C15:0. Galactose was detected as diagnostic sugar. Based on these chemotaxonomic results, 16S rRNA gene sequence analysis and DNA–DNA hybridization between strain CF5/2T and the type strains of Geodermatophilus saharensis, Geodermatophilus arenarius, Geodermatophilus nigrescens, Geodermatophilus telluris and Geodermatophilus siccatus, the isolates CF5/2T, CF5/1 and CF7/1 are proposed to represent a novel species, Geodermatophilus tzadiensis, with type strain CF5/2T = DSM 45416 = MTCC 11411 and two reference strains, CF5/1 (DSM 45415) and CF7/1 (DSM 45420).
Rock Biofilms
(2013)
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.
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.
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.
The order Chaetothyriales (Pezizomycotina, Ascomycetes) harbours obligatorily melanised fungi and includes numerous etiologic agents of chromoblastomycosis, phaeohyphomycosis and other diseases of vertebrate hosts. Diseases range from mild cutaneous to fatal cerebral or disseminated infections and affect humans and cold-blooded animals globally. In addition, Chaetothyriales comprise species with aquatic, rock-inhabiting, ant-associated, and mycoparasitic life-styles, as well as species that tolerate toxic compounds, suggesting a high degree of versatile extremotolerance. To understand their biology and divergent niche occupation, we sequenced and annotated a set of 23 genomes of main the human opportunists within the Chaetothyriales as well as related environmental species. Our analyses included fungi with diverse life-styles, namely opportunistic pathogens and closely related saprobes, to identify genomic adaptations related to pathogenesis. Furthermore, ecological preferences of Chaetothyriales were analysed, in conjuncture with the order-level phylogeny based on conserved ribosomal genes. General characteristics, phylogenomic relationships, transposable elements, sex-related genes, protein family evolution, genes related to protein degradation (MEROPS), carbohydrate-active enzymes (CAZymes), melanin synthesis and secondary metabolism were investigated and compared between species. Genome assemblies varied from 25.81 Mb (Capronia coronata) to 43.03 Mb (Cladophialophora immunda). The bantiana-clade contained the highest number of predicted genes (12 817 on average) as well as larger genomes. We found a low content of mobile elements, with DNA transposons from Tc1/Mariner superfamily being the most abundant across analysed species. Additionally, we identified a reduction of carbohydrate degrading enzymes, specifically many of the Glycosyl Hydrolase (GH) class, while most of the Pectin Lyase (PL) genes were lost in etiological agents of chromoblastomycosis and phaeohyphomycosis. An expansion was found in protein degrading peptidase enzyme families S12 (serine-type D-Ala-D-Ala carboxypeptidases) and M38 (isoaspartyl dipeptidases). Based on genomic information, a wide range of abilities of melanin biosynthesis was revealed; genes related to metabolically distinct DHN, DOPA and pyomelanin pathways were identified. The MAT (MAting Type) locus and other sexrelated genes were recognized in all 23 black fungi. Members of the asexual genera Fonsecaea and Cladophialophora appear to be heterothallic with a single copy of either MAT-1-1 or MAT-1-2 in each individual. All Capronia species are homothallic as both MAT1-1 and MAT1-2 genes were found in each single genome. The genomic synteny of the MAT-locus flanking genes (SLA2-APN2-COX13) is not conserved in black fungi as is commonly observed in Eurotiomycetes, indicating a unique genomic context for MAT in those species. The heterokaryon (het) genes expansion associated with the low selective pressure at the MAT-locus suggests that a parasexual cycle may play an important role in generating diversity among those fungi.
Simple, microbial biofilms have prevailed since life began on Earth. Biofilms form at the interfaces of solids with gases or liquids and have multiple effects on Substrate and element cycles. In geobiological terms, the most interesting microbial communities are those that form on solids exposed to air (subaerial). Microbial colonisers of the atmosphere-lithosphere interface include algae, cyanobacteria, fungi as well as heterotrophic bacteria and they have colonised virtually every rock surface throughout the entire geological history of the Earth. In addition to sequestering carbon, sub-aerial biofilms (SABS) actively participate in rock weathering. Rock-inhabiting SABs are the primary settlers on lava following volcanic eruptions and on rocks following the retreat of glaciers. SABs especially dominate hostile environments in which growth of higher Vegetation is restricted especially in deserts, polar- and alpine regions. SABs are the primary colonisers of lithospheric (e.g. rocks) and anthropogenic Substrates (buildings, monuments, solar panels, etc.). Life at the solid material/atmosphere interface influences and is affected by both the underlying Substrate and the microclimate surrounding it. Although subaerial life is ubiquitous, how SABs develop and importantly degrade underlying Substrates can only be clarified in well-controlled experiments that often involve simplified model Systems, So far, biofilm development on solar panels has been studied using; (i) metagenomics; (ii) in situ microscopy; and (iii) classical microbiological methods that are both qualitative and quantitative. Here we suggest that solar panel biofilms are accessible and highly relevant objects to study microbial ecology, geobiology and biodeterioration.
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.
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.
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.
Sub-aerial biofilms (SAB) are ubiquitous, self-sufficient microbial ecosystems found on mineral surfaces at all altitudes and latitudes. SABs, which are the principal causes of weathering on exposed terrestrial surfaces, are characterized by patchy growth dominated by associations of algae, cyanobacteria, fungi and heterotrophic bacteria. A recently developed in vitro system to study colonization of rocks exposed to air included two key SAB participants - the rock-inhabiting ascomycete Knufia petricola (CBS 123872) and the phototrophic cyanobacterium Nostoc punctiforme ATCC29133. Both partners are genetically tractable and we used them here to study weathering of granite, K-feldspar and plagioclase. Small fragments of the various rocks or minerals (1–6 mm) were packed into flow-through columns and incubated with 0.1% glucose and 10 μM thiamine-hydrochloride (90 μL min−1) to compare weathering with and without biofilms. Dissolution of the minerals was followed by: (i) analysing the degradation products in the effluent from the columns via Inductively Coupled Plasma Spectroscopy and (ii) by studying polished sections of the incubated mineral fragments/grains using scanning electron microscopy, transmission electron microscopy and energy dispersive X-ray analyses. K. petricola/N. punctiforme stimulated release of Ca, Na, Mg and Mn. Analyses of the polished sections confirmed depletion of Ca, Na and K near the surface of the fragments. The abrupt decrease in Ca concentration observed in peripheral areas of plagioclase fragments favored a dissolution-reprecipitation mechanism. Percolation columns in combination with a model biofilm can thus be used to study weathering in closed systems. Columns can easily be filled with different minerals and biofilms, the effluent as well as grains can be collected after long-term exposure under axenic conditions and easily analyzed.
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.
Life on the Rocks: A simple genetically tractable model system to study fungus-rock interactions
(2015)
Black microcolonial fungi (Ascomycetes from Arthonio-, Dothideo-, and Eurotiomycetes) are stress-tolerant and persistent dwellers of natural and anthropogenic extreme habitats. They exhibit slow yeast-like or meristematic growth, do not form specialized reproduction structures and accumulate the black pigment 1,8-dihydroxynaphthalene (DHN) melanin in the multilayered cell walls. To understand how black fungi live, survive, colonize mineral substrates, and interact with phototrophs genetic methods are needed to test these functions and interactions. We chose the rock inhabitant Knufia petricola of the Chaetothyriales as a model for developing methods for genetic manipulation. Here, we report on the expansion of the genetic toolkit by more efficient multiplex CRISPR/Cas9 using a plasmid-based system for expression of Cas9 and multiple sgRNAs and the implementation of the three resistance selection markers genR (geneticin/nptII), baR (glufosinate/bar), and suR (chlorimuron ethyl/sur). The targeted integration of expression constructs by replacement of essential genes for pigment synthesis allows for an additional color screening of the transformants. The black-pink screening due to the elimination of pks1 (melanin) was applied for promoter studies using GFP fluorescence as reporter. The black-white screening due to the concurrent elimination of pks1 and phs1 (carotenoids) allows to identify transformants that contain the two expression constructs for co-localization or bimolecular fluorescence complementation (BiFC) studies. The co-localization and interaction of the two K. petricola White Collar orthologs were demonstrated. Two intergenic regions (igr1, igr2) were identified in which expression constructs can be inserted without causing obvious phenotypes. Plasmids of the pNXR-XXX series and new compatible entry plasmids were used for fast and easy generation of expression constructs and are suitable for a broad implementation in other fungi. This variety of genetic tools is opening a completely new perspective for mechanistic and very detailed study of expression, functioning and regulation of the genes/proteins encoded by the genomes of black fungi.
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.
Plants and microorganisms, besides the climate, drive nitrogen (N) cycling in ecosystems. Our objective was to investigate N losses and N acquisition strategies along a unique ecosystem-sequence (ecosequence) ranging from arid shrubland through Mediterranean woodland to temperate rainforest. These ecosystems differ in mean annual precipitation, mean annual temperate, and vegetation cover, but developed on similar granitoid soil parent material, were addressed using a combination of molecular biology and soil biogeochemical tools. Soil N and carbon (C) contents, δ15N signatures, activities of N acquiring extracellular enzymes as well as the abundance of soil bacteria and fungi, and diazotrophs in bulk topsoil and rhizosphere were determined. Relative fungal abundance in the rhizosphere was higher under woodland and forest than under shrubland. This indicates toward plants' higher C investment into fungi in the Mediterranean and temperate rainforest sites than in the arid site. Fungi are likely to decompose lignified forest litter for efficient recycling of litter-derived N and further nutrients. Rhizosphere—a hotspot for the N fixation—was enriched in diazotrophs (factor 8 to 16 in comparison to bulk topsoil) emphasizing the general importance of root/microbe association in N cycle. These results show that the temperate rainforest is an N acquiring ecosystem, whereas N in the arid shrubland is strongly recycled. Simultaneously, the strongest 15N enrichment with decreasing N content with depth was detected in the Mediterranean woodland, indicating that N mineralization and loss is highest (and likely the fastest) in the woodland across the continental transect. Higher relative aminopeptidase activities in the woodland than in the forest enabled a fast N mineralization. Relative aminopeptidase activities were highest in the arid shrubland. The highest absolute chitinase activities were observed in the forest. This likely demonstrates that (a) plants and microorganisms in the arid shrubland invest largely into mobilization and reutilization of organically bound N by exoenzymes, and (b) that the ecosystem N nutrition shifts from a peptide-based N in the arid shrubland to a peptide- and chitin-based N nutrition in the temperate rainforest, where the high N demand is complemented by intensive N fixation in the rhizosphere.
Schwarze mikrokoloniale Pilze besiedeln zunehmend von Menschen geschaffene Habitate, wie schadstoffbelastete Böden, Statuen, Gebäudefassaden und Dächer. Sie verfärben und zersetzen die Oberflächen von anfälligen Materialien oder reduzieren die Lichtausbeute von Solaranlagen. Die Biologie dieser Pilze und ihre Relevanz für die Materialforschung stehen im Fokus unserer Studien an der Bundesanstalt für Materialforschung und -prüfung (BAM). Mit einer Kollektion schwarzer Pilze, die von Solaranlagen isoliert wurden, bringen wir klima- und materialrelevante Biodiversität in den Stammbaum des Lebens. Die Überlebensstrategien dieser Organismen versuchen wir mit molekularbiologischen und genetischen Untersuchungsansätzen zu entschlüsseln.
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.
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.