Identity and Structural Organization of a Microbial Community in a Model Drinking Water Distribution System

The aim of the present thesis is to determine the identity and structural organization of the indigenous bacteria present in a model drinking water distribution system. The study shows how unrecognized bacterial species in drinking water proliferate in two separate ecological compartments, in a biofilm on the pipe surface and free-swimming in the bulk water.

Initially, the development of a biofilm in a drinking water distribution system was analyzed with a simplified flow-cell system. The flow-cell set-up enabled a direct microscopic analysis of the biofilm. Microcolonies consisting of a mixed community including a- and â-Proteobacteria were seen with fluorescent in situ hybridization.

Furthermore, a variety of protozoans were present in the system, and some were attached to the microcolonies. A phylogenetic and physiological examination of isolates from two nonchlorinated drinking water distribution systems showed, that bacteria from the biofilm were on average able to utilize a higher number of substrates than strains from the bulk water.

Despite differences in taxonomic affiliation of the strains in the two analyzed systems, a parallel distribution of genetic and physiological diversity in the biofilm and bulk water was observed. However, in other environments, abundant bacteria have solely been detected using cultivation independent techniques.

Using cloning and sequencing of 16S rRNA fragments, it was demonstrated that bacteria from at least 12 phyla were present in the Danish model distribution system, including some of which never have been detected in drinking water. A bacterium affiliated to a nitrite oxidizer, Nitrospira, encompassed 39% of the bulk water and 25% of the biofilm community.

The close affiliation to Nitrospira suggested, that a large part of the population had an autotrophic metabolism. Bacteria affiliated to Acidobacterium and Planctomycetes were found in densities of up to 15%. An analysis of the community composition using terminal restriction fragment length polymorphism showed a correlation between the population profile and age of biofilm, separating the samples into a young (1 – 94 days) and an old biofilm (571 – 1093 days), whereas limited spatial variation in the biofilm was observed.

A more detailed analysis of 16S rRNA fragments demonstrated a unimodel relationship between the age and richness of the biofilm. Initially, a wide variety of cells were recruited from the bulk water colonizing the pipe surface and resulted in a species richness comparable to the water phase. This event was followed by growth of another bacterium from the phylum Nitrospirae, reaching 78% of the community by day 256.

Moreover, the bloom of this specie resulted in a drop in the relative richness. The biofilm entered a stable population from 500 days and on-wards, that was characterized by a higher diversity of bacteria. Visualization and subsequent quantification showed how the biofilm developed from an initial attachment of single cells, followed by formation of independent microcolonies reaching 30 mm in thickness, and finally to a looser structure with an average thickness of 14 mm and covering 76% of the surface.

The combination of different techniques illustrated the successional formation of a biofilm during a 3-year period in this model drinking water distribution system. A cluster analysis divided the young and old biofilm, and the bulk water communities into three separate groups. A detailed comparison between the communities in the biofilm and bulk water showed that certain species were solely found in microhabitat, whereas other species were present in both the biofilm and adjacent water phase.

Combined with the observed physiological difference between bacteria from the biofilm and bulk water, it appeared that many species had a primary habitat in either the biofilm or bulk water but that a dynamic exchange occurred between the communities.