DEVELOPMENT OF AN RIBOSOMAL-RNA-TARGETED OLIGONUCLEOTIDE PROBE SPECIFIC FOR THE GENUS ACINETOBACTER AND ITS APPLICATION FOR IN-SITU MONITORING IN ACTIVATED-SLUDGE

Enhanced biological phosphate removal in an anaerobic-aerobic activated sludge system has generally been ascribed to members of the genus Acinetobacter. A genus-specific 16S rRNA-targeted oligonucleotide probe was developed to investigate the role of acinetobacter spp. in situ. Nonisotopic dot blot hybridization to 66 reference strains, including the seven described Acinetobacter spp., demonstrated the expected probe specificity. Fluorescent derivatives were used for in situ monitoring of Acinetobacter spp, in the anaerobic and aerobic compartments of a sewage treatment plant with enhanced biological phosphate removal. Microbial community structures were further analyzed with oligonucleotide probes specific for the alpha, beta, or gamma subclasses of the class Proteobacteria, for the Cytophaga-Flavobacterium cluster, for gram-positive bacteria with a high G+C DNA content, and for all bacteria. Total cell counts were determined by 4',6-diamidino-2-phenylindole staining. In both the anaerobic and the aerobic basins, the activated sludge samples were dominated by members of the class Proteobacteria belonging to the beta subclass and by gram-positive bacteria with a high G+C DNA content. Acinetobacter spp. constituted less than 10% of all bacteria. For both basins, the microbial community structures determined with molecular techniques were compared with the compositions of the heterotrophic saprophytic microbiota determined with agar plating techniques. Isolates on nutrient-rich medium were classified by whole-cell hybridization with rRNA-targeted probes and fatty acid analysis. Cultivation on nutrient-rich medium favored the growth of members of the gamma subclass of Proteobacteria and selected against the growth of members of the beta subclass of Proteobacteria and gram-positive bacteria with a high G+C DNA content; 33% of the cultured bacteria from the anaerobic basin and 32% from the aeration basin were identified as Acinetobacter spp. The addition of small amounts of iron salts for chemical phosphate precipitation had no influence on the constitution of the microbial consortia. Enrichment of the return sludge with 20 mg of acetic acid per liter for 3 days significantly increased the relative abundance of gram-positive bacteria with a high G+C DNA content but had no effect on the numbers of Acinetobacter spp. The dominance of gram-positive bacteria with a high G+C DNA content and the presence of polyphosphate inclusions in these bacteria indicate that they may play a major role in biological phosphate removal.