Christine Müller-Renno

Cleaning of biomaterial surfaces: Protein removal by different solvents

The removal of biofilms or protein films from biomaterials is still a challenging task. In particular, for research investigations on real (applied) surfaces the reuse of samples is of high importance, because reuse allows the comparison of the same sample in different experiments. The aim of the present study was to evaluate the cleaning efficiency of different solvents (SDS, water, acetone, isopropanol, RIPA-buffer and Tween-20) on five different biomaterials (titanium, gold, PMMA (no acetone used), ceramic, and PTFE) with different wettability which were covered by layers of two different adsorbed proteins (BSA and lysozyme). The presence of a protein film after adsorption was confirmed by transmission electron microscopy (TEM). After treatment of the surfaces with the different solvents, the residual proteins on the surface were determined by BCA-assay (bicinchoninic acid assay). Data of the present study indicate that SDS is an effective solvent, but for several protein-substrate combinations it does not show the cleaning efficiency often mentioned in literature. RIPA-buffer and Tween-20 were more effective. They showed very low residual protein amounts after cleaning on all examined material surfaces and for both proteins, however, with small differences for the respective substrate-protein combinations. RIPA-buffer in combination with ultrasonication completely removed the protein layer as confirmed by TEM.

Cleaning of titanium substrates after application in a bioreactor

Plain and microstructured cp-titanium samples were studied as possible biofilm reactor substrates. The biofilms were grown by exposition of the titanium samples to bacteria in a flow cell. As bacteria the rod shaped gram negative Pseudomonas fluorescens and the spherical gram negative Paracoccus seriniphilus were chosen. Afterward, the samples were cleaned in subsequent steps: First, with a standard solvent based cleaning procedure with acetone, isopropanol, and ultrapure water and second by oxygen plasma sputtering. It will be demonstrated by means of x-ray photoelectron spectroscopy, fluorescence microscopy, and confocal laser scanning microscopy that oxygen plasma cleaning is a necessary and reliant tool to fully clean and restore titanium surfaces contaminated with a biofilm. The microstructured surfaces act beneficial to biofilm growth, while still being fully restorable after biofilm contamination. Scanning electron microscopy images additionally show, that the plasma process does not affect the microstructures. The presented data show the importance of the cleaning procedure. Just using solvents does not remove the biofilm and all its components reliably while a cleaning process by oxygen plasma regenerates the surfaces.

Novel Materials for Biofilm Reactors and their Characterization

The application of adherently growing microorganisms for biotechnological production processes is established, but it is still a niche technology with only a small economic impact. However, novel approaches are under development for new types of biofilm reactors. In this context, increasingly more microstructured metal surfaces are being investigated, and they show positive effects on the bacterial growth and the biofilm establishment. However, for comparison of the data, the different surface materials have to correspond in their different characteristics, such as wettability and chemical composition. Also, new materials, such as plastic composite supports, were developed. To understand the interaction between these new materials and the biofilm-producing microorganisms, different surface science methods have to be applied to reveal a detailed knowledge of the surface characteristics. In conclusion, microstructured surfaces show a high potential for enhanced biofilm growth, probably accompanied by an enhanced productivity of the microorganisms.

Albumin-lysozyme interactions: Cooperative adsorption on titanium and enzymatic activity

The interplay of albumin (BSA) and lysozyme (LYZ) adsorbed simultaneously on titanium was analyzed by gel electrophoresis and BCA assay. It was found that BSA and lysozyme adsorb cooperatively. Additionally, the isoelectric point of the respective protein influences the adsorption. Also, the enzymatic activity of lysozyme and amylase (AMY) in mixtures with BSA was considered with respect to a possible influence of protein-protein interaction on enzyme activity. Indeed, an increase of lysozyme activity in the presence of BSA could be observed. In contrast, BSA does not influence the activity of amylase.

Removing biofilms from stainless steel without changing surface properties relevant for bacterial attachment

The influence of oxygen (and argon) plasma cleaning and a base-acid cleaning procedure on stainless steel surfaces was studied. The main aim was to clean stainless steel samples from Paracoccus seriniphilus biofilms without changing the surface properties which are relevant for bacterial attachment to allow reuse in a biofilm reactor. It is shown that oxygen plasma cleaning, which very successfully removes the same kind of biofilm from titanium surfaces, is not suitable for stainless steel. It largely influences the surface chemistry by producing thick metal oxide layers of varying compositions and changing phenomenological surface properties such as wettability. A promising method without changing surface properties while cleaning satisfactorily is a combination of base and acid reagents at elevated temperature.

Adhesion forces of the sea-water bacterium Paracoccus seriniphilus on titanium: Influence of microstructures and environmental conditions

The bacterial attachment to surfaces is the first step of biofilm formation. This attachment is governed by adhesion forces which act between the bacterium and the substrate. Such forces can be measured by single cell force spectroscopy, where a single bacterium is attached to a cantilever of a scanning force microscope, and force-distance curves are measured. For the productive sea-water bacterium Paracoccus seriniphilus, pH dependent measurements reveal the highest adhesion forces at pH 4. Adhesion forces measured at salinities between 0% and 4.5% NaCl are in general higher for higher salinity. However, there is an exception for 0.9% where a higher adhesion force was measured than expected. These results are in line with zeta potential measurements of the bacterium, which also show an exceptionally low zeta potential at 0.9% NaCl. In the absence of macromolecular interactions, the adhesion forces are thus governed by (unspecific) electrostatic interactions, which can be adjusted by pH and ionic strength. It is further shown that microstructures on the titanium surface increase the adhesion force. Growth medium reduces the interaction forces dramatically, most probably through macromolecular bridging.

Electrostatic conditions define the 2D self-assembly of tomato bushy stunt viruses on solid surfaces

Plant viruses which are self-assembled on a substrate are interesting building blocks in nanobiotechnology, in particular, for the creation of 2D ordered structures. In this article, the self-assembly of different genetically modified types of the tomato bushy stunt virus spin-coated on pristine silicon was investigated by scanning force and scanning electron microscopy. Amino acid side chains were integrated in the capsids of the viruses by extending the coat protein with different charged amino acid clusters (tetra-aspartate-hexa-histidine, hexa-aspartate, or tetra-arginine-tags). The influence of the resulting electrostatic forces based on virus-virus and virus-surface interactions on the formation of self-assembled monolayers will be presented and discussed in the context of differences in surface coverage for different pH values. It could be shown that the largest surface coverage can be achieved when there is an attraction between the whole virus and the surface and only a minor repulsion between the viruses at a given pH.

Chloroperoxidase production by Caldariomyces fumago biofilms

Chloroperoxidase (CPO) is a versatile enzyme, which is secreted by the marine fungus Caldariomyces fumago (Leptoxyphium fumago). However, the application of the enzyme is hampered by its high price, which is due to the costly, labor‐intensive purification process. One challenge of the downstream process is the removal of a coproduced black pigment that forms a complex with the active enzyme. While strain development can be considered as an option to reduce the synthesis of the interfering pigment, the metabolism of the microorganism can be altered alternatively by using the biofilm growth mode of the fungus. The aim of this study was to reduce pigment formation during CPO synthesis. We investigated for the first time CPO production during C. fumago biofilm growth initiated through the presence of different microstructured stainless steel surfaces (material number: 1.4571; AISI 316Ti). CPO production by C. fumago was similar when grown as a biofilm or in suspension, whereas pigment formation was drastically reduced by cells grown on moderately structured surfaces (Ra = 0.13 ± 0.02 μm). The possibilities of biofilm growth for changing cell properties and for continuous fermentation are discussed.

Nanomechanical properties of the sea-water bacterium Paracoccus seriniphilus—A scanning force microscopy approach

The measurement of force-distance curves on a single bacterium provides a unique opportunity to detect properties such as the turgor pressure under various environmental conditions. Marine bacteria are very interesting candidates for the production of pharmaceuticals, but are only little studied so far. Therefore, the elastic behavior of Paracoccus seriniphilus, an enzyme producing marine organism, is presented in this study. After a careful evaluation of the optimal measurement conditions, the spring constant and the turgor pressure are determined as a function of ionic strength and pH. Whereas the ionic strength changes the turgor pressure passively, the results give a hint that the change to acidic pH increases the turgor pressure by an active mechanism. Furthermore, it could be shown, that P. seriniphilus has adhesive protrusions outside its cell wall.

Paracoccus seriniphilus adhered on surfaces: Resistance of a seawater bacterium against shear forces under the influence of roughness, surface energy, and zeta potential of the surfaces

Bacteria in flowing media are exposed to shear forces exerted by the fluid. Before a biofilm can be formed, the bacteria have to attach to a solid surface and have to resist these shear forces. Here, the authors determined dislodgement forces of single Paracoccus seriniphilus bacteria by means of lateral force microscopy. The first measurement set was performed on very flat glass and titanium (both as very hydrophilic samples with water contact angles below 20°) as well as highly oriented pyrolytic graphite (HOPG) and steel surfaces (both as more hydrophobic surfaces in the context of biological interaction with water contact angles above 50°). The different surfaces also show different zeta potentials in the range between -18 and -108 mV at the measurement pH of 7. The second set comprised titanium with different RMS (root mean square) roughness values from a few nanometers up to 22 nm. Lateral forces between 0.5 and 3 nN were applied. For Paracoccus seriniphilus, the authors found as a general trend that the surface energy of the substrate at comparable roughness determines the detachment process. The surface energy is inversely proportional to the initial adhesion forces of the bacterium with the surface. The higher the surface energy (and the lower the initial adhesion force) is, the easier the dislodgement of the bacteria happens. In contrast, electrostatics play only a secondary role in the lateral dislodgement of the bacteria and may come only into play if surface energies are the same. Furthermore, the surface chemistry (glass, titanium, and steel as oxidic surfaces and HOPG as a nonoxidic surface) seems to play an important role because HOPG does not completely follow the above mentioned general trend found for the oxide covered surfaces. In addition, the roughness of the substrates (made of the same material) is limiting the lateral dislodgement of the bacteria. All examined structures with RMS roughness of about 8-22 nm on titanium prevent the bacteria from the lateral dislodgement compared to polished titanium with an RMS roughness of about 3 nm.