In this study, we examined the long-term development of the overall structural morphology and community composition of a biofilm formed in a model drinking water distribution system with biofilms from 1 day to 3 years old. Visualization and subsequent quantification showed how the biofilm developed from an initial attachment of single cells through the formation of independent microcolonies reaching 30 m in thickness to a final looser structure with an average thickness of 14.1 m and covering 76% of the surface. An analysis of the community composition by use of terminal restriction fragment length polymorphisms showed a correlation between the population profile and the age of the sample, separating the samples into young (1 to 94 days) and old (571 to 1,093 days) biofilms, whereas a limited spatial variation in the biofilm was observed. A more detailed analysis with cloning and sequencing of 16S rRNA fragments illustrated how a wide variety of cells recruited from the bulk water initially attached and resulted in a species richness comparable to that in the water phase. This step was followed by the growth of a bacterium which was related to Nitrospira, which constituted 78% of the community by day 256, and which resulted in a reduction in the overall richness. After 500 days, the biofilm entered a stable population state, which was characterized by a greater richness of bacteria, including Nitrospira, Planctomyces, Acidobacterium, and Pseudomonas. The combination of different techniques illustrated the successional formation of a biofilm during a 3-year period in this model drinking water distribution system.

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In a model drinking water distribution system characterized by a low assimilable organic carbon content (<10 g/liter) and no disinfection, the bacterial community was identified by a phylogenetic analysis of rRNA genes amplified from directly extracted DNA and colonies formed on R2A plates. Biofilms of defined periods of age (14 days to 3 years) and bulk water samples were investigated. Culturable bacteria were associated with Proteobacteria and Bacteriodetes, whereas independently of cultivation, bacteria from 12 phyla were detected in this system. These included Acidobacteria, Nitrospirae, Planctomycetes, and Verrucomicrobia, some of which have never been identified in drinking water previously. A cluster analysis of the population profiles from the individual samples divided biofilms and bulk water samples into separate clusters (P ‫؍‬ 0.027). Bacteria associated with Nitrospira moscoviensis were found in all samples and encompassed 39% of the sequenced clones in the bulk water and 25% of the biofilm community. The close association with Nitrospira suggested that a large part of the population had an autotrophic metabolism using nitrite as an electron donor. To test this hypothesis, nitrite was added to biofilm and bulk water samples, and the utilization was monitored during 15 days. A first-order decrease in nitrite concentration was observed for all samples with a rate corresponding to 0.5 ؋ 10 5 to 2 ؋ 10 5 nitrifying cells/ml in the bulk water and 3 ؋ 10 5 cells/cm 2 on the pipe surface. The finding of an abundant nitrite-oxidizing microbial population suggests that nitrite is an important substrate in this system, potentially as a result of the low assimilable organic carbon concentration. This finding implies that microbial communities in water distribution systems may control against elevated nitrite concentrations but also contain large indigenous populations that are capable of assisting the depletion of disinfection agents like chloramines.Drinking water is an important resource all around the globe. Nevertheless, little research has focused on identifying the bacteria in water distribution systems or how treatment techniques alter the composition. In contrast, the bacteria present in wastewater treatment plants are very well characterized by a suite of advanced molecular techniques (40). The monitoring and identification of bacteria present in drinking water have primarily been achieved using a plating and isolation strategy. The majority of these culturable bacteria described in drinking water networks are included in the phylum Proteobacteria and to a lesser extent in the phyla Actinobacteria, Firmicutes, and Bacteriodetes (18,19,29,30,32). Some of the commonly detected genera include Pseudomonas, Caulobacter, Aeromonas, Acinetobacter, and Bacillus. As a result, a common perception is that Pseudomonas is the most abundant bacterial organism in supply systems, regardless of the water source.It is well known that the plate count technique significantly underestimates the total number of cells prese...

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As a representative member of the toluene-degrading population in a biofilter for waste gas treatment, Pseudomonas putida was investigated with a 16S rRNA targeting probe. The three-dimensional distribution of P. putida was visualized in the biofilm matrix by scanning confocal laser microscopy, demonstrating that P. putida was present throughout the biofilm. Acridine orange staining revealed a very heterogeneous structure of the fully hydrated biofilm, with cell-free channels extending from the surface into the biofilm. This indicated that toluene may penetrate to deeper layers of the biofilm, and consequently P. putida may be actively degrading toluene in all regions of the biofilm. Furthermore, measurements of growth rate-related parameters for P. putida showed reduced rRNA content and cell size (relative to that in a batch culture), indicating that the P. putida population was not degrading toluene at a maximal rate in the biofilm environment. Assuming that the rRNA content reflected the cellular activity, a lower toluene degradation rate for P. putida present in the biofilm could be estimated. This calculation indicated that P. putida was responsible for a significant part (65%) of the toluene degraded by the entire community.