Phosphorus (P) is one of the primary nutrients that must be removed from wastewater in order to prevent eutrophication in marine environments. Enhanced biological phosphorus removal (EBPR) is an activated sludge process that is widely accepted as the most economic and environmentally sustainable method of P removal when it is operated successfully. The group of bacteria that are responsible for P removal from wastewater are known as polyphosphate accumulating organisms (PAOs), which require sequential anaerobic and aerobic conditions for growth. PAOs are able to take up volatile fatty acids (VFAs) anaerobically and convert them to intracellular poly-β-hydroxyalkanoates (PHAs). The energy and reducing power for these processes are obtained through the hydrolysis of their intracellularly stored polyphosphate and glycogen. Aerobically, PAOs oxidise PHA to gain energy for growth, glycogen replenishment and P uptake. Phosphorus removal is achieved from the EBPR process through the wastage of excess sludge rich in polyphosphate. Deterioration in EBPR performance has been hypothesised to be linked to the proliferation of a group of bacteria known as the glycogen accumulating organisms (GAOs), which may compete in wastewater systems with PAOs. Like PAOs, GAOs are capable of taking up the often-limited VFA substrates from EBPR systems anaerobically. Unlike PAOs, however, GAOs do not contribute to P removal and they are therefore an undesirable organism in EBPR systems. The presence of GAOs would serve only to remove VFAs that would have ideally been metabolised by PAOs.
The presence of PAOs and GAOs in activated sludge may be examined microscopically using the fluorescence in situ hybridisation (FISH) technique. FISH probes have been designed to target "Candidatus Accumulibacter phosphatis" (henceforth referred to as Accumulibacter), the only currently identified PAO, and "Candidatus Competibacter phosphatis" (henceforth referred to as Competibacter), a known GAO. Accumulibacter and Competibacter have been shown to dominate many lab-scale EBPR cultures using quantitative FISH analysis.
This thesis focuses on the competition between PAOs and GAOs in EBPR systems. The presence and activity of PAOs and GAOs in lab-scale and full-scale systems are described through a combination of chemical analysis and microbiological techniques. This integrated approach produces a comprehensive analysis of the EBPR process, enabling a greater understanding of the effects of process factors such as carbon source and pH on the competition between these two groups of organisms. The main contributions from this thesis are described below:
Competibacter GAOs were shown to be present in full-scale EBPR plants and were demonstrated to consume VFAs in these systems. The minimisation of GAOs would result in more efficient carbon utilisation for phosphorus removal in EBPR systems, improve process stability, and could lower operational costs of the plant. This research was carried out in the early stages of the Ph.D. project, leading to the decision to focus this study on the competition between PAOs and GAOs.
A novel GAO belonging to the Alphaproteobacteria phylum was enriched with propionate as the sole carbon source. The anaerobic and aerobic biotransformations performed by this novel group of GAOs were characterised through examination of the solid, liquid and gas phase processes, which displayed a clear GAO phenotype. A metabolic model was proposed for the metabolism of propionate by GAOs, and was found to adequately describe the anaerobic stoichiometry observed through chemical analysis.
Accumulibacter PAOs were demonstrated to be able to utilise both acetate and propionate, and can readily switch between the two carbon sources. The anaerobic biochemical transformations associated with propionate uptake by PAOs were characterised through the use of a metabolic model. The model was shown to describe very well the anaerobic stoichiometry of an enriched culture of PAOs fed with propionate as the sole carbon source. A comparison between the metabolism of propionate and acetate by PAOs suggested that PAOs possess a similar preference towards the uptake of either acetate or propionate.
Carbon source was demonstrated to have a major impact on the competition between PAOs and GAOs. Propionate was shown to be a more favourable carbon source than acetate for reliable phosphorus removal as it provided PAOs an advantage over GAOs. Competibacter GAOs were unable to compete effectively with PAOs for propionate, while they were shown to be a strong competitor for acetate with PAOs. PAOs were also observed to compete more effectively than the Alphaproteobacteria GAOs for propionate uptake in a mixed culture containing both organisms. A consistently high and stable level of P removal was observed when propionate was fed as the sole carbon source, while an acetate fed system frequently showed periods of poor P removal. Thus, increasing the fraction of propionate through prefermentation or adding propionate as a supplementary carbon source may improve the reliability and efficiency of EBPR plants through the minimisation of GAOs. PAOs were also shown to have an advantage over GAOs in VFA uptake after a sudden change in the carbon source. Both Competibacter GAOs and Alphaproteobacteria GAOs were slower to respond to a change in carbon source as compared to PAOs. These different groups of GAOs have a preference towards the metabolism of either acetate or propionate, respectively, which suggests that each group of GAOs may be minimised in EBPR plants through external dosing of the non-preferred substrate.
A high operational pH was shown to provide PAOs an advantage over both the Competibacter GAOs and the Alphaproteobacteria GAOs. A pH increase from ~7 to ~8 resulted in a dramatic improvement in the phosphorus removal performance due to population shifts favouring PAOs over GAOs. The Alphaproteobacteria GAOs were observed to be a less effective competitor with PAOs at a high pH as compared to Competibacter. EBPR plant operation at a high pH level (~8) would likely minimise the proliferation of GAOs and improve the performance and efficiency of these systems.
An analytical method was optimised for quantification of one of the primary PHA fractions produced by PAOs or GAOs fed with propionate, known as poly-β-hydroxy-2-methylvalerate (PH2MV). It was shown that the PHA extraction method could be modified to yield improved recovery of all PHA fractions produced in EBPR systems fed with acetate or propionate.
This thesis shows that the minimisation of GAOs can indeed improve the level of phosphorus removal in EBPR systems by maximising the amount of VFA taken up by PAOs. The impact of carbon source and pH on the PAO-GAO competition was clearly demonstrated, and control strategies to select for PAOs over GAOs have been developed. The information obtained from this research may prove very useful towards the optimisation of full-scale EBPR plants.