In situ characterization of the microbial consortia active in two wastewater treatment plants

Activated sludge samples from a one-state-system of a municipal sewage plant and a sequencing batch reactor (SBR) treating dairy-sewage were characterized using 16S and 23S ribosomal RNA (rRNA) targeted oligonucleotide probes specific for defined phylogenetic groups of bacteria. Cell counts were determined by a combination of in situ hybridization with fluorescently-labelled oligonucleotide probes and epifluorescence microscopy. Around 80% of total cells, as determined with the DNA specific fluorochrome DAPI, hybridized with a Bacteria specific probe. This indicated that the majority of microscopically visualized cells were indeed physiologically active. The samples of the municipal sewage plant were dominated by proteobacteria belonging to the beta-subclass, whereas small cells hybridizing with a cytophaga-flavobacterium-specific probe were most abundant in the batch reactor. Comparison of in situ microbial community structures and the heterotrophic saprophyte flora (analysed with standard cultivation techniques) resulted in major discrepancies. The nutrient rich medium favoured growth of gamma-subclass proteobacteria (e.g. Acinetobacter sp.) which were only a minor constituents (< 10%) of both sludge consortia. Dominating groups like the beta-subclass proteobacteria, the cytophagaflavobacteria cluster and the Gram-positive bacteria with a high G + C content of DNA were suppressed. These results suggest that the application of in situ hybridization techniques can yield a more complete understanding of the microbial populations involved in the purification of sewage.