Erifyli Tsagkari

The potential of DWDS pipewall biofilms to shelter and propagate opportunistic pathogens is currently poorly understood. Here, we use an annular biofilm reactor approach to quantify the fate of the opportunistic pathogen Pseudomonas aeruginosa when introduced to a simulated DWDS environment. We found that P. aeruginosa was capable of swift attachment to surfaces and able to persist for up to 14 days under shear stress conditions. Further, we demonstrate that P. aeruginosa is capable of detachment/reattachment and mobilisation through the bulk water, potentially acting as a source of inoculum to drinking water.

The accumulation, growth, and re-mobilization of pathogens on the pipe walls in drinking water distribution systems are processes that affect the risk of exposure at the tap. We present a model that uses the Buckingham Pi theory to embody the physics of Pseudomonas aeruginosa accumulation and move within the system. We apply it to model experimental data from a biofilm annular reactor operated in conditions that are commensurate with the flow in DWDS. By calibrating the model for this benchtop system, we intend to identify the most important physical parameters for use in a simpler, more prudent model, for application in large-scale DWDS.

Water companies make efforts to reduce the risk of microbial contamination in drinking water. A widely used strategy is to introduce chlorine into the drinking water distribution system (DWDS). A subtle potential risk is that non-lethal chlorine residuals may select for chlorine resistant species in the biofilms that reside in DWDS. Here, we quantify the thickness, density, and coverage of naturally occurring multi-species biofilms grown on slides in tap water with and without chlorine, using fluorescence microscopy. We then place the slides in an annular rotating reactor and expose them to fluid-wall shears, which are redolent of those on pipe walls in DWDS. We found that biofilms in chlorine experiment were thicker, denser and with higher coverage than in non-chlorine conditions under all flow regimes and during incubation. This suggests that the formation and development of biofilms was promoted by chlorine. Surprisingly, for both chlorinated and non-chlorinated conditions, biofilm thickness, density and coverage were all positively correlated with shear stress. More differences were detected in biofilms under the different flow regimes in non-chlorine than in chlorine experiments. This suggests a more robust biofilm under chlorine conditions. While this might imply less mobilization of biofilms in high shear events in pipe networks, it might also provide refuge from chlorine residuals for pathogens.

There is growing evidence that individual bacteria sense and respond to changes in mechanical loading. However, the subtle responses of multispecies biofilms to dynamic fluid shear stress are not well documented because experiments often fail to disentangle any beneficial effects of shear stress from those delivered by convective transport of vital nutrients. We observed the development of biofilms with lognormally distributed microcolony sizes in drinking water on the walls of flow channels underflow regimes of increasing complexity. First, where regular vortices induced oscillating wall shear and simultaneously enhanced mass transport, which produced the thickest most extensive biofilms. Second, where unsteady uniform flow imposed an oscillating wall shear, with no enhanced transport, and where the biomass and coverage were only 20% smaller. Finally, for uniform steady flows with constant wall shear where the extent, thickness, and density of the biofilms were on average 60% smaller. Thus, the dynamics of shear stress played a significant role in promoting biofilm development, over and above its magnitude or mass transfer effects, and therefore, mechanosensing may prevail in complex multispecies biofilms which could open up new ways of controlling biofilm structure.

Bacterial motility is one important factor that affects biofilm formation. In drinking water there are key bacteria in aggregation, whose biology acts to enhance the formation of biofilms. However, it is unclear whether the motility of these key bacteria is an important factor for the interactions between bacteria in drinking water, and, subsequently, in the formation of aggregates, which are precursors to biofilms. Thus, the role of the motility of one of these key bacteria, the Methylobacterium strain DSM 18358, was investigated in the interactions between bacteria in drinking water. The motility of pure Methylobacterium colonies was initially explored; if it was affected by the viscosity of substrate, the temperature, the available energy and the type of substrate. Furthermore, the role of Methylobacterium in the interactions between mixed drinking water bacteria was investigated under the mostly favourable conditions for the motility of Methylobacterium identified before. Overall, the motility of Methylobacterium was found to play a key role in the communication and interactions between bacteria in drinking water. Understanding the role of the motility of key bacteria in drinking water might be useful for the water industry as a potential tool to control the formation of biofilms in drinking water pipes.

Biofilms are found at the inner surfaces of drinking water pipes and, therefore, it is essential to understand biofilm processes to control their formation. Hydrodynamics play a crucial role in shaping biofilms. Thus, knowing how biofilms form, develop and disperse under different flow conditions is critical in the successful management of these systems. Here, the development of biofilms after 4 weeks, the initial formation of biofilms within 10 h and finally, the response of already established biofilms within 24-h intervals in which the flow regime was changed, were studied using a rotating annular reactor under three different flow regimes: turbulent, transition and laminar. Using fluorescence microscopy, information about the number of microcolonies on the reactor slides, the surface area of biofilms and of extracellular polymeric substances and the biofilm structures was acquired. Gravimetric measurements were conducted to characterise the thickness and density of biofilms, and spatial statistics were used to characterise the heterogeneity and spatial correlation of biofilm structures. Contrary to the prevailing view, it was shown that turbulent flow did not correlate with a reduction in biofilms; turbulence was found to enhance both the initial formation and the development of biofilms on the accessible surfaces. Additionally, after 24-h changes of the flow regime it was indicated that biofilms responded to the quick changes of the flow regime. Overall, this work suggests that different flow conditions can cause substantial changes in biofilm morphology and growth and specifically that turbulent flow can accelerate biofilm growth in drinking water.

The structure of biofilms in drinking water systems is influenced by the interplay between biological and physical processes. Bacterial aggregates in bulk fluid are important in seeding biofilm formation on surfaces. In simple pure and co-cultures, certain bacteria, including Methylobacterium, are implicated in the formation of aggregates. However, it is unclear whether they help to form aggregates in complex mixed bacterial communities. Furthermore, different flow regimes could affect the formation and destination of aggregates. In this study, real drinking water mixed microbial communities were inoculated with the Methylobacterium strain DSM 18358. The propensity of Methylobacterium to promote aggregation was monitored under both stagnant and flow conditions. Under stagnant conditions, Methylobacterium enhanced bacterial aggregation even when it was inoculated in drinking water at 1% relative abundance. Laminar and turbulent flows were developed in a rotating annular reactor. Methylobacterium was found to promote a higher degree of aggregation in turbulent than laminar flow. Finally, fluorescence in situ hybridisation images revealed that Methylobacterium aggregates had distinct spatial structures under the different flow conditions. Overall, Methylobacterium was found to be a key strain in the formation of aggregates in bulk water and subsequently in the formation of biofilms on surfaces.