Community analysis of bacterial biofilms in a simulated recirculating cooling-water system by fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes

An open recirculating cooling-water system feeding a modified Robbin’s Device (MRD) with synthetic cooling water to simulate the environment of a recirculating industrial cooling-water system was set up. Both mild steel and Nylon® coupons were inserted to sample biofilms at regular intervals. The community structure was determined by in situ hybridization using fluorescently labelled 16S and 23S rRNA-targeted oligonucleotide probes specific for the beta-, gamma- and delta subclasses of the Proteobacteria, as well as probes specific for the genera Shewanella, Aeromonas and Pseudomonas. A Cy5 labelled eubacterial probe was used in conjunction with other probes to overcome the problems experienced with autofluorescence. Due to the weak fluorescent signals obtained, biofilm samples were treated with glucose, yeast extract and chloramphenicol before fixation, to allow the cell volume and ribosome count to increase while suppressing cell division. The predominant bacteria in biofilms in the recirculating cooling-water systems were found to be members of the beta Proteobacteria (ca 50% of total bacteria observed with EUB 338) and unidentified large cocci, whereas gamma Proteobacteria were only observed occasionally (ca 10% of total bacteria observed with EUB 338). At first no Pseudomonas were detected, but after the glucose, yeast extract and chloramphenicol treatment, ca 5% of the gamma Proteobacteria present were found to be Pseudomonas. Shewanella putrefaciens were present in low numbers (ca 10% of the gamma Proteobacteria present), although well distributed. We did not observe any sulphate-reducing bacteria or Aeromonas.