Isolierung und Charakterisierung von bakteriellen extrazellulären polymeren Substanzen aus Biofilmen
Microorganisms in biofilms are kept together by extracellular polymeric substances (EPS). The EPS are key molecules for the structure, function and organization of biofilms. Chemical and / or physical isolation methods are being used for the quantitative separation of EPS from biofilms. The yield of EPS depends on the method of isolation. Four different methods of EPS isolation were used in this work (separation by stirring and centrifugation, use of a cation exchange resin, extraction with formaldehyde and extraction with formaldehyde and NaOH) on pure culture biofilms of Pseudomonas aeruginosa and biofilms from sewage treatment systems. The isolation by stirring and centrifugation was suitable for pure culture biofilms. If calcium was present in the growth medium stirring and centrifugation alone was not sufficient. The isolation of EPS was successful with the cation exchange method. The method of choice for the isolation of EPS from environmental biofilms was the cation exchange method. EPS from pure culture biofilms of P. aeruginosa and P. fluorescens did not only consist of polysaccharides, but also of significant amounts of proteins. In environmental biofilms humic substances and DNA were found in addition to polysaccharides and proteins. Detailed studies of the EPS from P. aeruginosa showed, that the EPS consisted of 70 % (w/w) of alginate. Alginate showed a clear heterogeneity in relation to charge (acetylated and non-acetylated fraction) and molar mass. Neutral carbohydrates were not found in the EPS after total hydrolysis followed by thin layer chromatography. Proteins amounted to 28 % (w/w) of the EPS. It is assumable that this not only related to enzymes, but also structural proteins (e. g. lectins). Rhamnose lipids (mainly di-rhamno lipid) were also found in the EPS (small amount of 1 % (w/w)); these molecules may also play an important role in the development of the biofilm structure. By increasing the time of biofilm cultivation P. aeruginosa produced (related to cell number) more EPS (mainly alginate). The composition of the EPS was depending on the nutrient medium. In synthetic media high amounts of polysaccharides and almost no proteins (in contrast to rich media) were detected in the EPS. EPS of pure culture biofilms of P. fluorescens contained carbohydrates (57 % (w/w)) and proteins (28 % (w/w)). Acetyl groups (5 % (w/w)) and glucose and galactose after hydrolysis and thin layer chromatography were detected in the EPS. Possibly the exopolysaccharide of P. fluorescens is an acetylated galactoglucan. In the analyzed sludges of waste water treatment proteins followed by carbohydrates made up the main components of the EPS. Humic substances and small amounts of DNA were detected in these EPS. The EPS of aquatic biofilms contained large amounts of humic substances. Uronic acids were not detected in any analyzed environmental biofilm. Therefore acidic polysaccharides in these biofilms cannot play any role in the stabilization of biofilms by cross linking the EPS with multivalent cations. Instead of that, humic substances, nucleic acids and acidic proteins could be responsible for cross linking.