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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.
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.
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.
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.
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.
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.
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.
Materials that support natural biodiversity on their surfaces can compensate for human activities that have a negative impact on nature and thus contribute to a carbon-neutral and nature-positive world. Specifically designing bioreceptive materials which favor the growth of biofilms on their surface is an approach complementing conventional, macroscopic green façades. But what exactly characterizes a bioreceptive substrate and how do biofilm and substrate interact? How and why does a spontaneous colonization and the formation of biofilms take place? What are biofilms and how can they be established in a laboratory setting? How can this existing knowledge be transferred to the artificial stone concrete so that this material can be tuned to increase (or decrease) its bioreceptivity?
This review paper aims at summarizing the existing state of knowledge on bioreceptive concrete and pointing out inconsistencies and contradictions which can only be removed by more interdisciplinary research in the field.