Composition, dynamics and function of extracellular polymeric substances in drinking-water biofilms

Drinking water distribution systems as well as domestic plumbing systems are colonized by microbial biofilms. Under unfavourable conditions they may act as reservoirs for hygienically relevant microorganisms, posing a potential threat to human health. The aim of this study was the investigation of the formation, composition and function of drinking-water biofilms and their extracellular polymeric substances (EPS). The biofilms were grown on a synthetic elastomeric material, exposed to drinking water in different drinking water distribution systems as well as public plumbing systems made of copper. Characterization of biofilms and their EPS was carried out by microbiological, molecular biological and biochemical methods, which were adapted and optimized to meet the difficulty provided by the low amounts of biomass usually found in drinking water systems. The elastomeric material provided a suitable substratum for the cultivation of drinking-water biofilms. After 14 d of exposure, biofilm growth reached a quasi-stationary state, showing constant cell numbers in the range of 1.0 x 10^8 cells cm^-2 to 5.0 x 10^8 cells cm^-2. Culturability of biofilm cells was one order of magnitude lower compared to total cell numbers. Analysis by PCR-DGGE showed an increase in population diversity for the first 7 d of biofilm growth and from then on remained constant for up to 28 d. Comparison of band patterns of biofilms in different water systems showed variable microbial composition of drinking-water biofilms, with similarities of only 46 % to 63 %. EPS were isolated from drinking-water biofilms by application of a cation exchange resin (CER). Due to the low biomass yield this EPS isolation method was miniaturized and optimized, and compared to other commonly applied isolation methods including treatment by shaking (control), formaldehyde/NaOH, EDTA, or heat. The CER method showed significantly higher yields of EPS components compared to the control method, and, in contrast to chemical methods or heat, caused no damage to biofilm cells or interference with analyses. Proteins represented the main component, irrespective of biofilm age or origin, followed by carbohydrates and DNA. Protein and carbohydrate contents in the EPS increased continuously throughout the cultivation period of up to 28 d, while DNA showed an increase in concentration for the initial 11 d to 21 d of cultivation, from which on the concentration remained constant. The isolation yields of EPS constituents varied depending on the biofilm’s origin and showed a decreasing trend with increasing Cu content in the water phase. Similarly to protein production, also activity of the hydrolytic enzyme groups proteases, peptidases, α-/s-glucosidases, N-acetyl-s-D-glucosaminidases, lipases, esterases, and phosphatases increased with biofilm age, in particular once biofilms reached 14 d of age. EPS protein diversity was analyzed by two-dimensional gel electrophoresis and exhibited significant variability according to the biofilm’s origin. In the course of biofilm formation, diversity of EPS proteins increased for the first 14 d and decreased from then on, showing a lower amount of protein spots with high molecular weightes or isoelectric points. Analysis of protein spots by MALDI-TOF-MS identified proteins with metabolic, transport, or regulatory functions in the EPS of drinking-water biofilms. A few protein clusters, including efflux proteins, were only present in biofilms grown in copper plumbing systems. The incorporation of a hygienically relevant microorganism into drinking-water biofilms was examined. As a model microorganism of hygienical relevance, Pseudomonas aeruginosa was used to examine the hypothesis, that it can influence the composition of existing biofilms and their EPS. The incorporation of P. aeruginosa as detected by FISH showed the potential of drinking-water biofilms to harbor hygienically relevant microorganisms. An effect on the composition of drinking-water biofilms or their EPS was not observed. The results demonstrated the variability of drinking-water biofilms in terms of microbial populations and EPS composition in response to variations of conditions in different distribution systems and in particular in copper plumbing systems. Furthermore, this study demonstrated the dynamics of EPS components in the course of biofilm formation, indicating continuous changes to the EPS matrix induced by the constituent organisms. Drinking-water biofilms were shown to be another type of biofilms, in which proteins represent the main EPS component, followed by polysaccharides and DNA. EPS proteins in drinking-water biofilms exhibited metabolic, transport and regulatory functions.