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

Advanced Wastewater Management Centre, The University of Queensland, St Lucia 4072, Australia¹
Department of Geology and Geophysics, University of Wisconsin-Madison, Madison, WI 53706, USA²
School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK³

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

Abbreviations: CLSM, confocal laser scanning microscope/microscopy; COD, chemical oxygen demand; EBPR, enhanced biological phosphorus removal; FISH, fluorescence in situ hybridization; GAO, glycogen-accumulating organism; OTU, operational taxonomic unit; PAO, polyphosphate-accumulating organism; PHA, poly-ß-hydroxyalkanoate; SBR, sequencing batch reactor; VFA, volatile fatty acid

^b The GenBank accession numbers for the sequences reported in this paper are given in Methods.

^a Present address: Department of Environmental Science, Policy and Management, Hilgard Hall, University of California Berkeley, Berkeley CA 94720, USA.

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