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 ofAcinetobacter 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.

The structures of bacterial communities were studied in activated sludge samples obtained from the aerobic and anaerobic zones of a wastewater treatment plant showing enhanced phosphorous removal. Samples were analyzed by in situ hybridization with oligonucleotide probes complementary to selected regions of the 16S and 23S ribosomal RNA (rRNA) characteristic for defined phylogenetic entities (genera and larger groups). The microbial community structures revealed by molecular techniques were compared with the compositions of culturable bacterial communities, obtained from the characterization of 255 isolates from tryptone-soy (TS) agar and R2A agar. These isolates were characterized by 89 physiological tests and their cellular fatty acid patterns, and identified. Culture-dependent techniques indicated the following distribution: different Aeromonas spp. (2.7-8.3% on R2A agar; 45.0-63.7% on TS agar), Acinetobacter spp. (5.4-9.0% on R2A agar; 5.0-9.1% on TS agar), Pseudomonas spp. (up to 10% on R2A agar) and Shewanella putrefaciens (up to 3.0% on R2A agar), all members of the gamma subclass of Proteobacteria, were isolated most frequently. The relatively rare isolates of the beta subclass were identified as Acidovorax spp., Alcaligenes spp., and Comamonas spp.. The Gram-positive bacteria (high DNA G+C) were assigned mainly to Arthrobacter spp., Microbacterium spp., and Mycobacterium phlei. In order to assess the in situ abundance of the most frequently isolated genus, Aeromonas, two rRNA-targeted oligonucleotide probes were developed. The two

No abstract

To investigate the bacteria that are important to phosphorus (P) removal in activated sludge, microbial populations were analyzed during the operation of a laboratory-scale reactor with various P removal performances. The bacterial population structure, analyzed by fluorescence in situ hybridization (FISH) with oligonucleotides probes complementary to regions of the 16S and 23S rRNAs, was associated with the P removal performance of the reactor. At one stage of the reactor operation, chemical characterization revealed that extremely poor P removal was occurring. However, like in typical P-removing sludges, complete anaerobic uptake of the carbon substrate occurred. Bacteria inhibiting P removal overwhelmed the reactor, and according to FISH, bacteria of the β subclass of the class Proteobacteria other than β-1 or β-2 were dominant in the sludge (58% of the population). Changes made to the operation of the reactor led to the development of a biomass population with an extremely good P removal capacity. The biochemical transformations observed in this sludge were characteristic of typical P-removing activated sludge. The microbial population analysis of the P-removing sludge indicated that bacteria of the β-2 subclass of the class Proteobacteria and actinobacteria were dominant (55 and 35%, respectively), therefore implicating bacteria from these groups in high-performance P removal. The changes in operation that led to the improved performance of the reactor included allowing the pH to rise during the anaerobic period, which promoted anaerobic phosphate release and possibly caused selection against non-phosphate-removing bacteria.

Samples from a wastewater treatment plant were hybridized with fluorescein-labeled oligonucleotide probes specific for members of the domains Bacteria and Eucarya; the alpha, beta, and gamma subclasses of the class Proteobacteria; or the genus Acinetobacter. Subsequently, they were counterstained with the DNA-specific dye Hoechst 33342 and analyzed by flow cytometry. By quantifying forward angle light scatter and Hoechst-and probe-conferred fluorescence as measures for cell size, DNA content, and rRNA content, respectively, not only relative abundances but also assessments of general metabolic activity for each of these groups were obtained. Hybridizations with a positive control probe binding to all bacteria showed that in the activated-sludge samples examined, 70 to 80% of the Hoechst-stained cells could unambiguously be identified by this method. The majority of the detected cells (approximately 40%) were beta-subclass Proteobacteria. Flow cytometric and microscopic counts were in general agreement. Discrepancies were found in particular for those populations that occurred predominantly in flocs (alpha subclass of the Proteobacteria) or chains (Acinetobacter spp.). Although the dispersal of aggregates needs to be improved, flow cytometry combined with rRNA-based in situ probing appears to be a powerful tool for the rapid and highly automated analysis of the microbial communities in activated sludge.