PROBING ACTIVATED-SLUDGE WITH OLIGONUCLEOTIDES SPECIFIC FOR PROTEOBACTERIA - INADEQUACY OF CULTURE-DEPENDENT METHODS FOR DESCRIBING MICROBIAL COMMUNITY STRUCTURE

Bacterial community structures in activated sludge samples from aerati on tanks of a two-stage system with a high-load first stage and a low- load second stage were analyzed with oligonucleotide probes. The probe s were complementary to conserved regions of the rRNA of the alpha, be ta, and gamma subclasses of proteobacteria and of all bacteria. Group- specific cell counts were determined by in situ hybridization with flu orescent probe derivatives. Contributions of the proteobacterial subcl asses to total bacterial rRNA were quantified by dot blot hybridizatio n with digoxigenin-labeled oligonucleotides. The activated sludge samp les were dominated by proteobacteria from the alpha, beta, or gamma su bclass. These proteobacteria account for about 80% of all active bacte ria found in the activated sludge. For both samples the community stru ctures determined with molecular techniques were compared with the com position of the heterotrophic saprophyte flora isolated on nutrient-ri ch medium. Probes were used to rapidly classify the isolates and to di rectly monitor population shifts in nutrient-amended, activated sludge samples. The rich medium favored growth of gamma-subclass proteobacte ria (e.g., enterobacteria) and selected against beta-subclass proteoba cteria. The culture-dependent community structure analysis of activate d sludge produced partial and heavily biased results. A more realistic view will be obtained by using in situ techniques.