Bacterial community structures in activated sludge samples from aeration tanks of a two-stage system with a high-load first stage and a low-load second stage were analyzed with oligonucleotide probes. The probes were complementary to conserved regions of the rRNA of the alpha, beta, and gamma subclasses of proteobacteria and of all bacteria. Group-specific cell counts were determined by in situ hybridization with fluorescent probe derivatives. Contributions of the proteobacterial subclasses to total bacterial rRNA were quantified by dot blot hybridization with digoxigenin-labeled oligonucleotides. The activated sludge samples were dominated by proteobacteria from the alpha, beta, or gamma subclass. These proteobacteria account for about 80% of all active bacteria found in the activated sludge. For both samples the community structures determined with molecular techniques were compared with the composition of the heterotrophic saprophyte flora isolated on nutrient-rich medium. Probes were used to rapidly classify the isolates and to directly monitor population shifts in nutrient-amended, activated sludge samples. The rich medium favored growth of gamma-subclass proteobacteria (e.g., enterobacteria) and selected against beta-subclass proteobacteria. The culture-dependent community structure analysis of activated sludge produced partial and heavily biased results. A more realistic view will be obtained by using in situ techniques.

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.

No abstract

Wastewater treatment is a process of increasing importance in a world with an ever growing human population. Today, most wastewater treatment processes make use of the natural self‐purification capacity of aquatic environments which is the result of the presence and action of microbial communities. Consequently, wastewater treatment facilities are designed to maintain high densities and activities of those microorganisms that satisfy the various purification demands. The performance, at least of large plants, has to be constantly monitored and is subject to strict regulation. Nevertheless, malfunctions resulting in decreased purification efficacy are frequent. This has, over the decades, prompted many microbiologists to compare the structure, dynamics and function of these ‘good’ or ‘bad’, but always complex, microbial communities. Part of these studies was targeted to a basic understanding of the various processes, another part, however, deals with the monitoring of community structure as a means to direct the plant operation towards higher elimination rates and overall stability. Even though the last decade has seen a molecular revolution in microbiology, the standard methods for monitoring wastewater treatment plants still rely on the tools available to the researcher at the beginning of this century, the microscope and agar plates. It is the goal of this MiniReview to compare the potential of the newly available molecular methods with the old monitoring techniques.

The microbial community of a denitrifying sand filter in a municipal wastewater treatment plant was examined by conventional and molecular techniques to identify the bacteria actively involved in the removal of nitrate. In this system, denitrification is carried out as the last step of water treatment by biofilms growing on quartz grains with methanol as a supplemented carbon source. The biofilms are quite irregular, having a median thickness of 13 to 20 m. Fatty acid analysis of 56 denitrifying isolates indicated the occurrence of Paracoccus spp. in the sand filter. 16S rRNA-targeted probes were designed for this genus and the species cluster Paracoccus denitrificans-Paracoccus versutus and tested for specificity by whole-cell hybridization. Stringency requirements for the probes were adjusted by use of a formamide concentration gradient to achieve complete discrimination of even highly similar target sequences. Whole-cell hybridization confirmed that members of the genus Paracoccus were abundant among the isolates. Twenty-seven of the 56 isolates hybridized with the genus-specific probes. In situ hybridization identified dense aggregates of paracocci in detached biofilms. Probes complementary to the type strains of P. denitrificans and P. versutus did not hybridize to cells in the biofilms, suggesting the presence of a new Paracoccus species in the sand filter. Analysis using confocal laser scanning microscopy detected spherical aggregates of morphologically identical cells exhibiting a uniform fluorescence. Cell quantification was performed after thorough disruption of the biofilms and filtration onto polycarbonate filters. An average of 3.5% of total cell counts corresponded to a Paracoccus sp., whereas in a parallel sand filter with no supplemented methanol, and no measurable denitrification, only very few paracocci (0.07% of cells stained with 4,6-diamidino-2-phenylindole) could be detected. Hyphomicrobium spp. constituted approximately 2% of all cells in the denitrifying unit and could not be detected in the regular sand filter. This clear link between in situ abundance and denitrification suggests an active participation of paracocci and hyphomicrobia in the process. Possible selective advantages favoring the paracocci in this habitat are discussed.