Biofilm Formation and Control in a Novel Warm Water Distribution System By Paul Lewis Waines Abstract Investigations were carried out to assess biofilm formation within a model warm water distribution system (test rig) under a variety of conditions. Analysis methods included ATP-/ culture-based analysis, SEM and confocal microscopy. Molecular-based community analysis was carried out using PCR/DGGE. High pH (9.53-10.08), induced by the presence of a sacrificial anode within the water heater, had a profound inhibitive effect on the culturability of biofilm bacteria on copper (Cu) pipe within the test rig. Concurrent investigations into the effect of stagnation (varied periods of non-flushing) appeared to contradict the widely held view that stagnation is conducive to biofilm formation, with greater flushing frequencies resulting in increased biofilm. It was concluded that a higher frequency of nutrient-delivering events were largely responsible for this and that in systems where lengthier stagnation periods were employed, factors such as low oxygen and reduced nutrient levels inhibited biofilm formation on previously uncontaminated Cu pipe. Thermal purging (TP) over a 28 day period of 30 second, 12 hourly flushing at 41 °C and three-daily one minute purging with 70 °C water resulted in a 99% reduction in the culturability of biofilm bacteria on both Cu and LLDPE. However, confocal microscopical analysis of bacterial numbers indicated that 25.06% (Cu) and 21.55% (LLDPE) of the initial bacterial population remained viable. A large proportion of non-viable biofilm bacteria were also observed. Further work is therefore required in order to optimize TP within the test rig. Biofilm formation on a range of different materials; Cu, stainless steel, PEX, and EPDM, showed significantly greater biofilm development on EPDM in comparison to the other materials. Preliminary investigations of LLDPE and tap outlet fittings showed that laminar flow outlet fittings may act as reservoirs for the development and subsequent dissemination of biofilm. Molecular bacterial community structural studies of test rig biofilms clearly showed that biofilm community composition was significantly affected by both temporal and environmental factors, and varied at points within the same system. Sequencing did not provide a great insight into the composition of the bacterial communities within the test rig, and further work is required to gain a more complete picture of bacterial community diversity within the test rig. These studies show that biofilm formation within the test rig is greatly influenced by a wide variety of factors. The test rig’s unique design necessitates a cautionary approach when making comparisons with, for example, larger water distribution systems

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