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 activate d sludge system has generally been ascribed to members of the genus Ac inetobacter. A genus-specific 16S rRNA-targeted oligonucleotide probe was developed to investigate the role of acinetobacter spp. in situ. N onisotopic dot blot hybridization to 66 reference strains, including t he 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. Mic robial 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-po sitive bacteria with a high G+C DNA content, and for all bacteria. Tot al cell counts were determined by 4',6-diamidino-2-phenylindole staini ng. In both the anaerobic and the aerobic basins, the activated sludge samples were dominated by members of the class Proteobacteria belongi ng to the beta subclass and by gram-positive bacteria with a high G+C DNA content. Acinetobacter spp. constituted less than 10% of all bacte ria. For both basins, the microbial community structures determined wi th molecular techniques were compared with the compositions of the het erotrophic saprophytic microbiota determined with agar plating techniq ues. Isolates on nutrient-rich medium were classified by whole-cell hy bridization with rRNA-targeted probes and fatty acid analysis. Cultiva tion on nutrient-rich medium favored the growth of members of the gamm a subclass of Proteobacteria and selected against the growth of member s of the beta subclass of Proteobacteria and gram-positive bacteria wi th a high G+C DNA content; 33% of the cultured bacteria from the anaer obic basin and 32% from the aeration basin were identified as Acinetob acter spp. The addition of small amounts of iron salts for chemical ph osphate precipitation had no influence on the constitution of the micr obial consortia. Enrichment of the return sludge with 20 mg of acetic acid per liter for 3 days significantly increased the relative abundan ce of gram-positive bacteria with a high G+C DNA content but had no ef fect on the numbers of Acinetobacter spp. The dominance of gram-positi ve bacteria with a high G+C DNA content and the presence of polyphosph ate inclusions in these bacteria indicate that they may play a major r ole in biological phosphate removal.