Glycogen-accumulating organisms in laboratory-scale and full-scale wastewater treatment processes

Advanced Wastewater Management Centre, The University of Queensland, St Lucia 4072, Australia 1 Department of Geology and Geophysics, University of Wisconsin-Madison, Madison, WI 53706, USA 2 School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK 3 Author for correspondence: Linda L. Blackall. Tel: +61 7 3365 4645. Fax: +61 7 3365 4699. e-mail: blackall{at}biosci.uq.edu.au Laboratory-scale sequencing batch reactors (SBRs) as models for wastewater treatment processes were used to identify glycogen-accumulating organisms (GAOs), which are thought to be responsible for the deterioration of enhanced biological phosphorus removal (EBPR). The SBRs (called Q and T), operated under alternating anaerobic–aerobic conditions typical for EBPR, generated mixed microbial communities (sludges) demonstrating the GAO phenotype. Intracellular glycogen and poly-ß-hydroxyalkanoate (PHA) transformations typical of efficient EBPR occurred but polyphosphate was not bioaccumulated and the sludges contained 1·8% P (sludge Q) and 1·5% P (sludge T). 16S rDNA clone libraries were prepared from DNA extracted from the Q and T sludges. Clone inserts were grouped into operational taxonomic units (OTUs) by restriction fragment length polymorphism banding profiles. OTU representatives were sequenced and phylogenetically analysed. The Q sludge library comprised four OTUs and all six determined sequences were 99·7% identical, forming a cluster in the - Proteobacteria radiation. The T sludge library comprised eight OTUs and the majority of clones were Acidobacteria subphylum 4 (49% of the library) and candidate phylum OP10 (39% of the library). One OTU (two clones, of which one was sequenced) was in the - Proteobacteria radiation with 95% sequence identity to the Q sludge clones. Oligonucleotide probes (called GAOQ431 and GAOQ989) were designed from the - Proteobacteria clone sequences for use in fluorescence in situ hybridization (FISH); 92% of the Q sludge bacteria and 28% of the T sludge bacteria bound these probes in FISH. FISH and post-FISH chemical staining for PHA were used to determine that bacteria from a novel - Proteobacteria cluster were phenotypically GAOs in one laboratory-scale SBR and two full-scale wastewater treatment plants. It is suggested that the GAOs from the novel cluster in the - Proteobacteria radiation be named ‘ Candidatus Competibacter phosphatis’. Keywords: GAOs, fluorescence in situ hybridization (FISH), wastewater treatment, EBPR Abbrevia