Quantification of functional groups in activated sludge by microautoradiography

Different functional groups of bacteria in activated sludge from a Danish municipal wastewater treatment plant were investigated and quantified on the basis of their ability to take up acetate under different electron acceptor conditions. The number of bacteria in the different functional groups was quantified by microautoradiography or by fluorescence in situ hybridization (FISH). Uptake of radiolabeled acetate was tested under conditions where oxygen, nitrate, ferric iron, and sulfate served as electron acceptors and under methanogenic conditions. Ammonia-oxidizing bacteria and nitrite-oxidizing bacteria were enumerated by applying oligonucleotide probes (FISH). 80% of the total DAPI count hybridised with a mixture of bacterial probes. Most baceria were able to take up acetate with oxygen or nitrate as electron acceptors (74 and 71% of all DAPI-stained bacteria, respectively). The very similar numbers indicate that the alternating aerobic and anoxic conditions in the wastewater treatment plant investigated favoured the presence of facultative aerobic and denitrifying heterotrophic bacteria. The number of bacteria able to take up acetate under anaerobic conditions was around 8% of the total DAPI count. About half of these were able to take up acetate and store it and were thus probably phosphate-accumulating organisms. The remaining 4% of anaerobic acetate-consuming bacteria were mainly iron reducers. By incubating the sludge samples with specific inhibitors against sulfate reduction and methanogenic activity, it was found that sulfate reducers and methanogenic bacteria constituted approximately 1% and less than 0.5% of the total DAPI count, respectively.