Nitrogen (N) and phosphorus (P) are the key nutrients causing eutrophication in waterways. Biological nutrient removal (BNR) is widely accepted as one of the most economic and environmental friendly processes to remove nitrogen and phosphorus from the wastewater. A more complete understanding of the mechanisms of BNR is highly important to improve BNR plant design and operations, enabling these systems to meet the ever stricter effluent discharge standards with minimum costs.
This thesis focuses on the role of intracellular storage products in biological nutrient removal. The crucial role of intracellular carbon storage products such as poly-hydroxyalkanoates (PHAs) and glycogen in the metabolisms of polyphosphate-accumulating organisms (PAOs) has been investigated by many researchers in the last decade or so. In more recent years, the central role of intracellular carbon storage products in the metabolisms of glycogen-accumulating organisms (GAOs), the competitor to PAOs in Enhanced Biological Phosphorus Removal (EBPR) systems, has also been recognised, although the study on the GAO metabolic pathways has been quite limited to date. Furthermore, it has been recognised that PAOs, in this case called denitrifying PAOs or DPAOs, are able to take up phosphate using nitrate as the electron acceptor, achieving simultaneous N and P removal. Here again, the intracellular carbon storage products, serving as both the carbon and energy sources, play an indispensable role.
A detailed study of the formation and usage of intracellular storage products by PAOs, DPAOs, GAOs and DGAOs (denitrifying GAOs, only a postulated group of organisms at the start of the project) was carried out. The study involved the use of lab-scale sequencing batch reactors (SBRs) running with different operational conditions that were believed to selectively enrich the desired groups of microorganisms. The carbon, nitrogen and phosphorus conversions were monitored by measuring, with both off-line lab analysis and on-line sensors, the relevant compounds in the solid, liquid and gas phases. This made it possible to establish complete mass balances for carbon, oxygen and nitrogen, as well as redox balances. The integration of the measured data with modelling and simulation tools allowed to achieve a significantly improved understanding of the metabolisms of the above organisms, in particular in relation to the roles of intracellular carbon storage products.
The main contributions of this research are:
• A complete metabolic model of GAOs was developed. The aerobic GAO model is the first model that characterises the aerobic activities of these organisms at a detailed level. Experimental validation showed that the model could predict the anaerobic and aerobic behaviour of GAOs very well. During model validation, it was found that at pH 7 the maximum anaerobic acetate uptake rate of GAOs was lower than that reported for PAOs. On the other hand, the net biomass yield for GAOs was about 9% higher than for PAOs. It was hypothesised that the above two differences could play crucial roles in the competition between PAOs and GAOs in EBPR systems.
• For the first time, DGAOs were successfully enriched under anaerobic/anoxic conditions. It was found that the anaerobic behaviour of DGAOs could be predicted well by the anaerobic GAO model initially proposed in the literature and amended in this thesis. It was observed that the final product of denitrification by DGAOs was mainly nitrous oxide (N2O) rather than N2. The data strongly suggested that the N2O production was caused by the inhibition of the nitrous oxide reductase due to an elevated level of nitrite accumulation during denitrification.
• A model-based data analysis method was developed and validated for the calculation of acetate uptake rates by PAOs and GAOs in a mixed culture. This method forms a powerful tool for the studies of PAO and GAO competition. A simplified method using only the acetate and glycogen data and the relevant stoichiometry was also developed and validated.
• It was further shown that PAOs and DPAOs are the same organisms: Accumulibacter phosphatis. DPAOs possess enzymes allowing them to accomplish aerobic enhanced biological phosphorus removal (EBPR) metabolism, while PAOs require a lag time to assumingly synthesise the necessary enzymes for anoxic EBPR metabolism. Potentially the conventional anaerobic-aerobic EBPR systems could be modified to optimise the use of anaerobic-anoxic EBPR, which can achieve denitrification and P removal simultaneously.
• Simultaneous nitrification, denitrification and phosphorus removal was demonstrated to be possible in a simple, single reactor process using anaerobic and low dissolved oxygen (0.5 mg/L) aerobic periods. However, off-gas analysis found that the final denitrification product was mainly nitrous oxide (N2O) rather than N2. Further experimental results demonstrated that nitrogen removal was via nitrite instead of nitrate. These experiments also showed that DGAOs rather than DPAOs were carrying out the denitrification in the reactor.
This thesis demonstrates that the intracellular storage products form a strong link between nitrogen and phosphorus removal processes in biological nutrient removal. This link needs to be explored towards simultaneous nitrogen and phosphorus removal.