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Biochemische und physiologische Charakterisierung der extrazellulären Matrix eines Modellbiofilms
(2018)
An der Grenzschicht zwischen einer festen Oberfläche und der umgebenden Luft wachsende subaerische Biofilme (SAB) zeichnen sich durch eine erhöhte Toleranz gegenüber extremen Umweltbedingungen und dem Eintrag von Bioziden aus. Dieser Schutz vor äußeren Umwelteinflüssen wird vornehmlich durch den Beitrag von extrazellulären polymeren Substanzen (EPS) und Pigmenten, wie Melanin und Carotinoiden, vieler unterschiedlicher Organismen gewährleistet. Deren Synthese wird wiederum durch intrazelluläre Botenstoffe reguliert. Das Cyanobakterium Nostoc punctiforme und der mikrokoloniale Pilz Knufia petricola, als Partner eines etablierten SAB-Modells und Vertreter zweier typischer Organismengruppen in SAB, wurden genutzt, um Botenstoffe und Pigmente genetisch zu manipulieren und die Biofilmmatrix dieser Mutanten strukturell zu untersuchen. In dieser Arbeit konnten erstmals die EPS beider Organismen extrahiert und die Struktur der extrazellulären Polysaccharide beschrieben werden. Daneben wurden die extrazellulären Polysaccharide von K. petricola Wildtyp mit denen verschiedener Pigmentmutanten verglichen. Das Fehlen des Schutzpigmentes Melanin führte zu einer ausgeprägteren extrazellulären Matrix in den Biofilmen. Gleichzeitig änderte sich die Struktur der extrazellulären Polysaccharide. Während der Wildtyp zu ~80% ein α-Glucan und zu ~20% ein α/β-Galaktomannan sekretierte, war der Anteil des Galaktomannans bei den Melaninmutanten erhöht. Das Ausschalten der Carotinoid-Synthese hatte jedoch keinen Einfluss auf die Beschaffenheit der extrazellulären Polysaccharide. Ein deutlich komplexeres extrazelluläres Polysaccharid aus acht verschiedenen Monosaccharid-Einheiten bildete N. punctiforme. Durch die Überproduktion des bakteriellen sekundären Botenstoffes c-di-GMP konnte zudem ein grundlegender Einfluss auf die Reaktion des Cyanobakteriums gegenüber externen Signalen und die damit verbundene Zelldifferenzierung gezeigt werden. Ein artifiziell erhöhtes c-di-GMP-Level in den Zellen führte zur Ausbildung eines sessilen Lebensstils durch Hemmung der Differenzierung motiler Hormogonien und vermehrte Produktion von EPS. Die Struktur der extrazellulären Polysaccharide wurde dadurch nicht verändert. Neben der strukturellen Analyse konnten die Veränderungen der extrazellulären Matrix beider Organismen zusätzlich durch mikroskopische Methoden visualisiert werden. Beide Organismen steuern komplexe Polymere, deren Produktion maßgeblich mit intrazellulären Faktoren verknüpft ist, zu der extrazellulären Matrix des Modellbiofilms bei.
Fungal biofilms on materials: describing and modelling growth of the black fungus Knufia petricola
(2023)
Fungi that grow as biofilms are associated with clinical settings as well as various cases of material fouling and material damage. Black fungi as biofilm formers have been rarely studied so far. Their conspicuous dark pigmentation, EPS production, adhesion capabilities and adaptations to stresses allow black fungi to develop biofilms on materials under harsh conditions. For example, rock-inhabiting black fungi withstand sun irradiation and dehydration and are therefore ubiquitous on arid surfaces like solar panels and marble monuments.
To understand and control their ability to colonise and deteriorate materials, one should assess and model black fungi’s growth patterns. But so far, no mathematical model has been developed to describe their growth. Knufia petricola A95, representing rock-inhabiting fungi from Chaetothyriales, is genetically amenable and can serve as a model for biofilm studies in black fungi. The primary objective of this project is to develop a growth model for K. petricola A95 which will enable to define and predict material colonisation of black fungi.
Dedicated experimental work with K. petricola will allow the quantitative assessment of the impact of environmental conditions (e.g. pH, nutrients, etc.) on the growth behaviour at the biofilm and single cells level. Data which will be used to validate and develop an individual-based model (based on the iDynoMICS modelling platform) that explains how fungal biofilms form, colonise materials, and cause deterioration.
Thus far, research has been conducted on the impact of different concentrations and sources of major elements (e.g. C, N, …), as well as trace elements (e.g. Cu, Mg, …), on the colony shape and biomass of Knufia petricola A95 biofilms. To study the behaviour of single cells, the length of the cell cycle in different growth media has been determined via the combined use of microfluidic devices and confocal microscopy.
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.
Microbiologically influence corrosion (MIC) has become a big concern due the increased usage of different metals by our society. Microorganisms can use metal as an electron donor, causing unpredictable but serious damages. Nowadays it is known that besides sulfate reducing bacteria (SRB), other microorganisms including acetogens, iron oxidizers and methanogens can also induce MIC. Current studies related to methanogen-induced MIC (MI-MIC) mainly focused on environmental isolates from the oil and gas industry (e.g. Methanococcus maripaludis) with industrial materials e.g. iron. However, MI-MIC can occur in many other environments as well, including the oral cavity. Methanobrevibacter oralis is a methanogen isolated from the human oral cavity and was found more frequently in patients suffering from peri-implantitis/periodontitis. Titanium-implants removed from those patients have also showed clear signs of corrosion. The aim of our study is to establish and analyze corrosion potentials of dental metals (e.g. titanium) by oral methanogens. Periodontal pockets samples from patients suffering from periodontitis/peri-implantitis were taken for methanogenic and SRB enrichments. Stainless steel, pure titanium or Ti-6Al-4V alloy was used for corrosion studies. Corrosion rates and methane production were measured using weight-loss method and gas chromatography, respectively. Metal surfaces were visualized with scanning electron microscopy. Microbial communities in the dental pockets of healthy people and patients will be compared using 16S rRNA amplicon sequencing. Overall, this is the first study investigating the susceptibility of different dental implant materials to corrosion using human-related Archaea. The outcomes of this study can be further explored for a variety of clinical applications.
Since the early 19th century microorganisms were studied on their capabilities of causing microbiologically influenced corrosion (MIC) of metals. The most studied ones are sulfate-reducing bacteria (SRB), but others can corrode metals as well, e.g. acid-producing bacteria or methanogenic archaea (MA). However, these studies were mostly focused on metals related to the petroleum industry but metals for other industries, e.g. dentistry, are also susceptible to corrosion. The inert Titanium (Ti) is often used as an implant material, but it is a base metal. The formation of a passivating oxide layer allows Ti to be corrosion resistant at normal conditions.
Nonetheless, scanning electron microscope images on dental implants from patients with acute peri-implantitis showed clear signs of corrosion. Currently, the corrosion mechanism of dental implants is unknown, but many indications suggest that oral microorganisms, including MA (Methanobrevibacter oralis) and SRB (Desulfomicrobium orale), could be involved.
To determine if MA or SRB can corrode Ti (pure Ti or Ti-6Al-4V alloy), corrosion rate, methane and sulfide concentrations were analyzed. Electrical potential measurements using in-house developed electrochemical cells indicated a potential change on Ti in the presence of a corrosive MA strain compared to an abiotic control.
Microbial composition comparison will be analyzed using samples from dental pockets of 150 infected patients by considering the quality of the implant and 50 healthy people by means of amplicon sequencing. Enrichments and isolation of pure cultures from the dentals samples are also examined for their corrosion behavior. Overall, this is the first study investigating the susceptibility of dental implant material to corrosion using human related MA.
Microbially influenced corrosion (MIC) of iron is usually attributed to sulfate-reducing microorganisms (SRM) which act upon the metal by the reactiveness of hydrogen sulfide, and by withdrawal of the available electrons (Fe → Fe²⁺ + 2e⁻ ; E° = 0.47 V) in electrical contact through surface attachment. Also methanogenic archaea are supposed to cause MIC. Because they do not produce hydrogen sulfide, withdrawal of electrons may be their main corrosive mechanism; however, mechanistic details and kinetics of the overall process are poorly understood. Precipitation of siderite (4Fe + 5HCO₃⁻ + 5H⁺ → 4FeCO₃ + CH₄ + 3H₂O) can lead to an insulating layer on the metal surface and lower the corrosion rate. Still, the extent of FeCO₃ precipitation may be significantly influenced by environmental conditions such as pH and advective processes.
To investigate the corrosive potential of methanogens, we studied strains isolated from marine sediments (Methanococcus maripaludis 14266, 2067, Methanobacterium-affiliated strain IM1), crude oil tanks (Methanococcus maripaludis Mic1c10, KA1) and the oral cavity (Methanobrevibacter oralis) in a closed (batch) culture, and in a sand-packed flow-through cell with pH control and simulation of a fluctuating environment. Results indicate that the rates of iron corrosion due to coupled methanogenesis (up to 0.3 mm/yr) are comparable to that caused by SRM. Surface analyses of the metal showed severe pitting. Such knowledge and deeper understanding also from an electrokinetic point of view may not only provide further models in microbial electrophysiology, but also contribute to mitigation strategies in MIC.
Microbiologically influenced corrosion (MIC) of iron is usually attributed to sulfate-reducing microorganisms, either chemically (formation of hydrogen sulfide) or electrically (direct electron uptake). Methanogenetic Archaea are also known to be involved in iron corrosion, forming a multi-species biofilm on corroding metallic structures. However, mechanistic details and kinetics of the overall process in methanogen-induced MIC are poorly understood.
Any surface in the environment acts as hotspot for microbial attachment and activity. These biofilms represent the interface between humans and the environment.
While in the past biofilms were often seen as disturbance, we now start to understand the enormous potential of beneficial biofilms. They can be used in a broad range of applications and are sources for new microorganisms and traits. After all, biofilms represent a great example for a collaborative lifestyle.
The Microplastic Microbiome
(2024)
Microplastics represent man-made and newly emerging surfaces in our ecosystems, where they interact with microorganisms. The ecosystem in focus of this presentation will be the aquatic environment. It will be portrayed, which microorganisms use microplastics as a habitat, how environmental factors shape this colonization, and why the biodegradation of plastics in the ocean is an overall unlikely process. We will also discuss whether potentially pathogenic microorganisms use microplastics as a raft. Finally, possible adaptation mechanisms of plastic-colonizing microorganisms will be presented, such as the production of photoreactive molecules. The microplastic microbiome has a large potential to harbor so far unknown species with curious traits, representing an exciting research topic for the future.
Bacteria are ubiquitous and colonize all types of surfaces, including those in close proximity to humans, such as skin, food, and everyday objects. This raises the question of whether their presence represents a problem to be mitigated or a potential source of benefit to be harnessed, thereby stimulating scientific inquiry into the role of surface-associated bacteria in diverse domains ranging fromhuman health to industrial biotechnology. Aim: The objective of this project is to explore the impact of modifying surface topography on bacterial adhesion behavior. By manipulating the physical characteristics of the substrate, the attachment and detachment dynamics of bacteria can potentially be modified, leading to novel strategies for controlling bacterial colonization in various applications, such as medical devices. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were tested on LIPSS-covered Fused Silica samples.
We demonstrate a 2D platform based on high contrast wetting patterns suitable for miniaturized microbiological assays.
In principal, superhydrophilic spots are surrounded by a superhydrophobic surface area. The special structure of the superhydrophilic functional surface ensures that liquids, e.g. bacterial suspensions or biocide solutions, spread immediately and evenly on this surface without passing the wetting boundary. This feature allows a homogenous distribution of bacteria or chemical substances on well defined lateral dimensions. The superhydrophilic spots may also serve as substrate for bacterial biofilms. Due to the high wetting contrast and the fabrication process, it is possible to minimize the test areas as well as their distance to each other.
We demonstrate the fabrication process of the high wetting contrast platform and also present a microbiological assay as an application example. Advantages of this platform are the use of low volumes and its potential of automated analysis.
Bacterial adhesion on surfaces of medical, water and food applications may lead to infections, water or food spoilage and human illness. In comparison to traditional static and macro flow chamber assays for biofilm formation studies, microfluidic chips allow in situ monitoring of biofilm formation under various flow regimes, have better environment control and smaller sample requirements.
In this work, a novel microfluidic platform is developed to investigate biofilm adhesion under precisely controlled bacteria concentration, temperature, and flow conditions. This platform central unit is a single-inlet microfluidic flow cell with a 5 mm wide chamber designed and tested to achieve ultra-homogenous flow in the central area of chamber. Within this area, defined microstructures are integrated that will disturb the homogeneity of the flow, thus changing bacterial adhesion pattern.
Here we present the monitoring of bacterial biofilm formation in a microfluidic chip equipped with a microstructure known as micro-trap. This feature is based on a 3D bacteria trap designed by Di Giacomo et al. and successfully used to sequester motile bacteria.
At first, fluorescent particles similar in size to Escherichia coli (E. coli) are used to simulate bacteria flow inside the flow cell and at the micro-trap. The turbulences induced by the trap are analyzed by imaging and particle tracking velocimetry (PTV). Secondly, the model strain E. coli TG1, ideal and well described for biofilm studies, is used to analyze biofilm formation in the micro-trap. Therefore, a stable fluorescent strain E. coli TG1-MRE-Tn7-141 is constructed by using Tn7 transposon mutagenesis according to the method described by Schlechter et al. Sequestering of E. coli cells within the micro-trap was followed using epifluorescence microscopy.
The novel microfluidic platform shows great potential for assessment of bacterial adhesion under various flow regimes. The performance of structural feature with respect to the generation of turbulences that promote or reduce bacterial adhesion can be systematically examined.
The combination of flow analysis and fluorescent strain injection into the microfluidic chip shows that the micro-trap is useful for capturing bacteria at defined positions and to study how flow conditions, especially micro-turbulences, can affect biofilm formation. It represents a powerful and versatile tool for studying the relation between topography and bacteria adhesion.