Dynamics of biofilm formation in drinking water: Phylogenetic affiliation and metabolic potential of single cells assessed by formazan reduction and in situ hybridization

Fluorescence-labeled oligonucleotide probes were applied, combined with in situ reduction of the fluorochrome 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), to describe the development of bacterial density, phylogenetic diversity and bacterial metabolic activity during the formation of drinking water biofilms. Polyethylene and glass surfaces exposed to drinking water in a modified Robbins device were rapidly colonized by a biofilm community of phylogenetically diverse prokaryotes, and cell density of the biofilm community was strictly controlled by grazing eukaryotic organisms. In situ hybridization with group-specific rRNA-targeted oligonucleotide probes revealed the following: (i) the prevalence of bacteria belonging to the beta-subclass of Proteobacteria within the bacterial biofilm populations; (ii) differences in the population composition, assessed by phylogenetic probes, depended on the surface properties of the substrata; (iii) the influence of water retention time on variations in population structure; and (iv) the presence of bacteria belonging to the family Legionellaceae associated with grazing protozoa. The metabolic potential of bacteria was assessed during biofilm formation using fluorescence signals after in situ hybridization and the reduction of the redox dye CTC as an indicator of respiratory activity. Respiratory activity and ribosome content of adherent bacterial cells decreased continuously during the early stages of the biofilm. After 35 days the percentage of CTC-reducing cells stabilized at 30%, and the amount of hybridized cells stabilized at 55%, of the initial cell number. To ascertain the amount of dormant, but potentially active cells, we established a new method, defined as probe active counts (PAC). Biofilms were incubated with a mixture of appropriate carbon sources and an antibiotic preventing bacterial cell division, followed by the determination of metabolic activity by in situ hybridization. By this approach the percentage of hybridized cells could be increased from 50% to 80% of total bacterial cell counts in the oligotrophic drinking water biofilms.