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 wit h in situ reduction of the fluorochrome 5-cyano-2,3-ditolyl tetrazoliu m chloride (CTC), to describe the development of bacterial density, ph ylogenetic diversity and bacterial metabolic activity during the forma tion of drinking water biofilms. Polyethylene and glass surfaces expos ed to drinking water in a modified Robbins device were rapidly coloniz ed by a biofilm community of phylogenetically diverse prokaryotes, and cell density of the biofilm community was strictly controlled by graz ing eukaryotic organisms. In situ hybridization with group-specific rR NA-targeted oligonucleotide probes revealed the following: (i) the pre valence of bacteria belonging to the beta-subclass of Proteobacteria w ithin the bacterial biofilm populations; (ii) differences in the popul ation composition, assessed by phylogenetic probes, depended on the su rface properties of the substrata; (iii) the influence of water retent ion time on variations in population structure; and (iv) the presence of bacteria belonging to the family Legionellaceae associated with gra zing protozoa. The metabolic potential of bacteria was assessed during biofilm formation using fluorescence signals after in situ hybridizat ion and the reduction of the redox dye CTC as an indicator of respirat ory activity. Respiratory activity and ribosome content of adherent ba cterial cells decreased continuously during the early stages of the bi ofilm. After 35 days the percentage of CTC-reducing cells stabilized a t 30%, and the amount of hybridized cells stabilized at 55%, of the in itial cell number. To ascertain the amount of dormant, but potentially active cells, we established a new method, defined as probe active co unts (PAC). Biofilms were incubated with a mixture of appropriate carb on sources and an antibiotic preventing bacterial cell division, follo wed by the determination of metabolic activity by in situ hybridizatio n. By this approach the percentage of hybridized cells could be increa sed from 50% to 80% of total bacterial cell counts in the oligotrophic drinking water biofilms.