Monitoring of biofilms grown on differentially structured metallic surfaces using confocal laser scanning microscopy.

Imaging of biofilms on opaque surfaces is a challenge presented to researchers especially considering pathogenic bacteria, as those typically grow on living tissue, such as mucosa and bone. However, they can also grow on surfaces used in industrial applications such as food production, acting as a hindrance to the process. Thus, it is important to understand bacteria better in the environment they actually have relevance in. Stainless steel and titanium substrata were line structured and dotted surface topographies for titanium substrata were prepared to analyze their effects on biofilm formation of a constitutively green fluorescent protein (GFP)-expressing Escherichia coli strain. The strain was batch cultivated in a custom built flow cell initially for 18 h, followed by continuous cultivation for 6 h. Confocal laser scanning microscopy (CLSM) was used to determine the biofilm topography. Biofilm growth of E. coli GFPmut2 was not affected by the type of metal substrate used; rather, attachment and growth were influenced by variable shapes of the microstructured titanium surfaces. In this work, biofilm cultivation in flow cells was coupled with the most widely used biofilm analytical technique (CLSM) to study the time course of growth of a GFP-expressing biofilm on metallic surfaces without intermittent sampling or disturbing the natural development of the biofilm.

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.

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.

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.

Analyzing the influence of microstructured surfaces on the lactic acid production of Lactobacillus delbrueckii lactis in a flow-through cell system.

Microorganisms growing in biofilms might be possible biocatalysts for future biotechnological production processes. Attached to a surface and embedded in an extracellular polymeric matrix, they create their preferred environment and form robust cultures for continuous systems. With the objective of implementing highly efficient processes, productive biofilms need to be understood comprehensively. In this study, the influence of microstructured metallic surfaces on biofilm productivity was researched. To conduct this study, titanium and stainless steel sheets were polished, micromilled, as well as coated with particles. Subsequently, the metal sheets were exposed to the lactic acid producing Lactobacillus delbrueckii subsp. lactis under laminar and homogeneous flow conditions in a custom-built flow cell. A proof-of-concept showed that biofilm formation in the system only occurred on the designated substratum. Following a 24-h batch cultivation for primary biofilm development, the culture was continuously provided with glucose containing medium. As different experimental series have indicated, the process resulted to be stable for up to eleven days. Primary metabolite productivity averaged around 6-7 g/(L h). Interestingly, the productivity was shown to be affected neither by the type of metal, nor by the applied microstructures. Nevertheless, a higher dry biomass weight determined on micro-milled substratum indicates a complementary differentiation of biofilm components in future experiments.