Since the isolation of bacteria in pure culture, investigations of microbial physiology have focussed on model microorganisms grown in pure culture. However, in order to understand complex environmental processes, there is a need to investigate mixed microbial communities. This is true for enhanced biological phosphorus removal (EBPR), a wastewater treatment process that results in the enrichment of polyphosphateaccumulating organisms (PAOs) and glycogen non-polyphosphate accumulating organisms (GAOs). PAOs and GAOs are key groups of physiologically distinct microorganisms that can proliferate in EBPR processes. PAOs drive the EBPR process, removing Pi from the wastewater, whereas GAOs negatively impact the process. In situ studies of Defluviicoccus in full-scale plants. Putative GAOs from the Alphaproteobacteria, Defluviicoccus (including Defluviicoccus vanus) were studied in full-scale EBPR plants to determine their distribution, abundance and ecological physiology (ecophysiology). Quantitative fluorescence in situ hybridization (FISH) demonstrated that Defluviicoccus were generally in low abundance, however in one plant surveyed, Cluster 2 Defluviicoccus comprised 9% of Bacteria. FISH combined with microautoradiography (MAR) revealed that both Cluster 1 and Cluster 2 Defluviicoccus were capable of taking up a narrow range of substrates including acetate, propionate, and pyruvate under anaerobic and aerobic conditions. Formate, butyrate, ethanol and several other organic carbon substrates were not taken up. Cluster 2 Defluviicoccus demonstrated a phenotype consistent with the current metabolic model for GAOs - anaerobic assimilation of acetate as polyhydroxyalkanoates (PHAs) with concomitant glycogen catabolism and aerobic consumption of PHA. Evidence was provided that these GAOs are likely to be unable to denitrify. The PAO, Accumulibacter and other GAOs (Competibacter) co-existed in two full-scale plants with Cluster 2 Defluviicoccus, but in both plants, the latter organisms were more abundant. Thus Cluster 2 Defluviicoccus can be relatively abundant and could be carbon (C) competitors with PAOs and other GAOs in EBPR plants. Bioenergetic models for Accumulibacter and Defluviicoccus. Investigations of acetate and inorganic phosphate (Pi) uptake in enrichments of Accumulibacter and acetate uptake in enrichments of Defluviicoccus were carried on lab-scale enrichment cultures and bioenergetic models were proposed. For both enrichments anaerobic acetate uptake assays in the presence of the protonophore, carbonyl cyanide m-chlorophenylhydrazone or the electrical potential () uncoupler valinomycin, indicated that acetate is likely to be taken up by a permease driven by the component of the proton motive force (pmf). Further investigation with the sodium ionophore monensin, suggested that anaerobic acetate uptake by Defluviicoccus may in part be sodium-dependent. Results of this study suggest that Accumulibacter generate a pmf for anaerobic acetate uptake by efflux of protons in symport with Pi through an inorganic phosphate transport system. In contrast, this study suggests that the anaerobic pmf in Defluviicoccus is generated by an efflux of protons across the cell membrane by the fumarate respiratory system, or by extrusion of sodium ions via decarboxylation of methylmalonyl-CoA. Aerobic Pi uptake by the Accumulibacter enrichment was strongly inhibited in the presence of an ATPase inhibitor (N, N’-dicyclohexylcarbodiimide), suggesting that the phosphate specific transport (Pst) system is important even under relatively high concentrations of Pi. Acetate permease activity in Accumulibacter and Defluviicoccus may play an important role in the competition for acetate in the often acetate limited EBPR process. Activity of a highvelocity Pst system in Accumulibacter may further explain its ability to compete strongly in EBPR. Anaerobic central metabolism in Accumulibacter. This study integrated in situ structure-function techniques, community biochemical measurements, metagenomic and transcriptomic analysis to study the physiology of Accumulibacter enriched in EBPR sludge communities. Anaerobic acetate uptake and assimilation as PHA in Accumulibacter was confirmed using FISH-MAR and post-FISH chemical staining. The effect of inhibitors on acetate uptake and storage polymer metabolism in the Accumulibacter enrichment was consistent with C flux through the glycolytic pathway and the glyoxylate cycle. Bioinformatic analysis of sequenced strains of Accumulibacter suggested that this PAO may interconvert intermediates of glycolysis and the glyoxylate cycle via malate-pyruvate and oxaloacetate-phosphenolpyruvate cycling. Investigation of gene expression in Accumulibacter demonstrated anaerobic activity of aconitase, isocitrate lyase, succinate dehydrogenase and cytochrome b/b6. A fusion protein including a novel cytochrome b/b6 complex likely facilitates energetically unfavourable anaerobic C flux through succinate dehydrogenase in Accumulibacter by pushing electrons uphill to more electronegative electron carriers. Physiological data from this study is interpreted in light of previous metagenomic information from enriched EBPR sludge communities and integrated with previous metabolic models for PAOs to develop a model for anaerobic central metabolism in Accumulibacter. Anaerobic central metabolism in Defluviicoccus. A lab-scale GAO enrichment culture dominated by Defluviicoccus was investigated to determine central metabolic pathways involved in anaerobic formation of PHA, a key carbon storage polymer essential for survival and proliferation of microorganisms in EBPR systems. Glycogen levels decreased under anaerobic conditions in the enrichment culture. However, no decrease in glycogen was observed in the presence of the glyceraldehyde-3-phosphate dehydrogenase inhibitor iodoacetate, inferring that glycogen is catabolized anaerobically in Defluviicoccus. FISH-MAR and post-FISH chemical staining supported the idea that acetate is converted to PHA in Defluviicoccus under anaerobic conditions. Anaerobic acetate uptake rates and PHA formation in the presence of metabolic inhibitors of central metabolic pathways in the Defluviicoccus enrichment culture was determined. Inhibition of isocitrate lyase by 3-nitropropionate and itaconate, indicated that C is likely to be channelled through the glyoxylate cycle in Defluviicoccus. Inhibitors of aconitase and succinate dehydrogenase suggested that aconitase but not succinate dehydrogenase was active, providing further support for the role of the glyoxylate cycle in these GAOs. The observed fumarate reductase inhibitor effect on PHA production indicated reduction of fumarate to succinate and the operation of the reductive branch of the TCA cycle. Intracellular polyhydroxyvalerate was measured in the Defluviicoccus enrichment and was likely produced by degradation of succinate generated by the glyoxylate cycle or fumarate reduction to propionyl-CoA followed by condensation with acetyl-CoA via a methylmalonyl-CoA intermediate.
| Identifer | oai:union.ndltd.org:ADTP/252445 |
| Creators | Luke Burow |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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