Molecular Characterization of Microbial Populations in Full-Scale Activated Sludge Plants and Bioaugmentation Sites

Abstract

The application of molecular biology techniques to the study of wastewater treatment and bioaugmentation was demonstrated in three studies. In the first study, full scale activated sludge treatment plants in North Carolina were surveyed to (1) determine the extent of filamentous bulking and foaming, and (2) relate these problems to the microbial community structures in the activated sludge reactors. Oligonucleotide probes targeting the rRNA of the major sublasses of the Proteobacteria, the mycolic acid containing actinomycetes (mycolata) and Sphaerotilus natans were used in quantitative hybridizations with samples from sixteen full scale plants. The survey results showed that 88% and 63% of plants in North Carolina have experienced bulking and foaming, respectively. No statistically significant correlations between the frequency and severity of foaming and the levels of any of the microbial groups were observed. However, several of the plants that had the most severe foaming problems had high levels of mycolata and alpha Proteobacteria. Bulking in the sampled plants is probably not caused by Sphaerotilus natans but by other filaments, or other floc characteristics. The sampling protocol raised questions about possible change between collection and analysis, and a second study was designed to determine optimal storage conditions. Quantitative hybridizations with the same set of probes used in the first study showed that treatment with chloramphenicol proved best in minimizing change over time in wastewater samples. However, storage at room temperature was also a viable storage option. In the third study, molecular methods were used to detect the bacterium Bacillus DA33 in augmented wastewater and soil samples. A probe targeting the 16S rRNA of this organism was designed and characterized for use in quantitative membrane hybridizations. Because a unique target sequence was not available within the 16S rRNA, another probe, this time targeting the 16S-23S intergenic spacer region, was designed and characterized. Hybridization results showed that levels of Bacillus DA33 were higher in soil than in wastewater. In several bioaugmented soil and wastewater samples, Bacillus DA33 had higher levels than in non-bioaugmented samples, but the results were mixed in other cases. Molecular methods allow us to analyze environmental samples in more detail. Integrating these methods in future studies of wastewater treatment and bioaugmentation should provide more information that will help improve design and operation.