1. ¹Advanced Water Management Centre, The University of Queensland, St Lucia, Queensland, Australia
2. ²School of Molecular and Microbial Sciences, The University of Queensland, St Lucia, Queensland, Australia

Correspondence: LL Blackall, Australian Institute of Marine Science, PMB 3, Townsville MC, Queensland 4810, Australia. E-mail: l.blackall@aims.gov.au

³Current address: Australian Institute of Marine Science, Queensland, Australia.

Received 12 April 2008; Accepted 14 April 2008; Published online 11 September 2008.

Abstract

Enhanced biological phosphorus removal (EBPR) communities protect waterways from nutrient pollution and enrich microorganisms capable of assimilating acetate as polyhydroxyalkanoate (PHA) under anaerobic conditions. Accumulibacter, an important uncultured polyphosphate-accumulating organism (PAO) enriched in EBPR, was investigated to determine the central metabolic pathways responsible for producing PHA. Acetate uptake and assimilation to PHA in Accumulibacter was confirmed using fluorescence in situ hybridization (FISH)-microautoradiography and post-FISH chemical staining. Assays performed with enrichments of Accumulibacter using an inhibitor of glyceraldehyde-3-phosphate dehydrogenase inferred anaerobic glycolysis activity. Significant decrease in anaerobic acetate uptake and PHA production rates were observed using inhibitors targeting enzymes within the glyoxylate cycle. Bioinformatic analysis confirmed the presence of genes unique to the glyoxylate cycle (isocitrate lyase and malate synthase) and gene expression analysis of isocitrate lyase demonstrated that the glyoxylate cycle is likely involved in PHA production. Reduced anaerobic acetate uptake and PHA production was observed after inhibition of succinate dehydrogenase and upregulation of a succinate dehydrogenase gene suggested anaerobic activity. Cytochrome b/b₆ activity inferred that succinate dehydrogenase activity in the absence of external electron acceptors may be facilitated by a novel cytochrome b/b₆ fusion protein complex that pushes electrons uphill to more electronegative electron carriers. Identification of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase genes in Accumulibacter demonstrated the potential for interconversion of C₃ intermediates of glycolysis and C₄ intermediates of the glyoxylate cycle. Our findings along with previous hypotheses from analysis of microbiome data and metabolic models for PAOs were used to develop a model for anaerobic carbon metabolism in Accumulibacter.