Biodiversity of Polyphosphate Accumulating Bacteria in Eight WWTPs with Different Modes of Operation

Enhanced biological phosphorous removal (EBPR) from wastewater has been successfully used for more than three decades and is considered to be an environmentally friendly wastewater-treatment process. Biologically, this process is realized by incorporation of phosphate as polyphosphate (polyP) granules in activated sludge bacteria. Important groups of bacteria responsible for P removal have been identified, but the full microbial diversity involved in this process is still unknown. This paper reports on the microbial composition of activated sludge communities in eight wastewater-treatment plants (WWTPs) with different sizes and modes of operation. The polyphosphate accumulating organisms (PAOs) within this complex biocenosis were identified by fluorescent dye staining and classified by in situ hybridization techniques. Of the bacteria in the aerobic basin, 5-13% contained polyP granules. In addition, flow cytometry was used to quantify PAOs after tetracycline staining and to separate these cells. The phylogenetic affiliation of the sorted PAOs was identified by cloning and sequencing. Both workflows showed similar outcomes. The majority of PAOs in all plants were Betaproteobacteria (22%), Actinobacteria (21%), and Alphaproteobacteria (12%), with differences in the relative abundance. In addition, Bacteroidetes (12%) were detected in the clone libraries, especially Haliscomenobacter, which should be considered further with regard to its influence on the EBPR process. The denaturing gradient gel electrophoresis analyses of sorted PAOs revealed a diverse community composition of Betaproteobacteria, Actinobacteria, and Rhodocyclales in the WWTPs. PAOs were present in EBPR and non-EBPR WWTPs, and no correlation in their abundance and phylogenetic composition to the mode of operation was revealed. This study shows that specific PAO communities existed in the various WWTPs, probably favored by the respective wastewater composition, including so far unvalued PAOs species, but their active contribution in the EBPR process remains to be investigated.