Ces travaux de thèse porte sur l'étude d'un procédé de traitement des eaux usées intitulé "anammox". C'est un procédé biologique reposant sur le métabolisme d'un groupe de bactéries du même nom permettant l'épuration de l'azote. Ces travaux développent une méthodologie pour leur identification et leur culture et aboutissent à la mise en application de bio-réacteur de traitement à l'échelle du laboratoire. / This work focuses on the study of a wastewater treatment process entitled "anammox". It is a biological process based on the metabolism of a group of bacteria of the same name allowing the purification of nitrogen. This work develops a methodology for their identification, their culture and for the implementation of bioreactor treatment at the laboratory scale.

Partial nitritation combined with the anaerobic ammonium oxidation (Anammox) process offers a way of replacing the conventional nitrogen removal process of nitrification-denitrification, lowering the need for oxygen and chemical input, as well as reducing the production of sludge. However, as a by-product of the biochemical reaction driven by anammox bacteria, it produces nitrate-nitrogen (NO3- - N) (16-26% nitrogen removed), which is problematic. Microbial desalination cells (MDCs) are a promising technology capable of converting biodegradable organics into electricity (by electroactive bacteria), providing for simultaneous desalination, and wastewater treatment. Despite being a promising technology, MDCs have limitations. The first-proof of-concept of MDC was demonstrated using acetate as the organic source, expensive platinum as a catalyst, and ferricyanide as an electron acceptor in the cathode that makes MDC costly, environmentally unfriendly, and unsustainable. This research investigated the integration of the anammox and nitration processes in MDCs as a long-term biocatalyst/biocathode for sustainable and energy-efficient nitrogen removal and electricity generation. A series of experiments were designed and performed to evaluate the performance of the anammox process as a biocatalyst in MDCs. The results concluded that the anammox process can be used as a biocatalyst to accept electrons in MDCs producing 444 mW/m3 of power density and 84% of ammonium nitrogen removal. Furthermore, the concept of using a one-stage nitritation anammox process as a biocathode in MDC was evaluated and produced a maximum power output of 1007 mW/m3. Two configurations of anammox MDCs (anaerobic-anammox cathode MDC (AnAmmoxMDC) and nitritation-anammox cathode MDC (NiAmoxMDC) were compared with an air cathode MDC (CMDC), operated in fed-batch mode. The NiAmoxMDC showed better performance in terms of power production and nitrogen removal. The co-existence of aerobic ammonium oxidizing bacteria (AOB) and anammox bacteria in the same biocathode of single-stage NiAmoxMDC concluded the resource-efficient wastewater treatment. Furthermore, two-stage nitritation anammox as a biocathode in MDC was evaluated and proved to be energy-efficient bioelectrochemical wastewater treatment by producing 1500 mW/m3 (300 mW/m2) of maximum power output. This research provides the first proof of concept that nitritation-anammox biocathode can provide a sustainable and energy-efficient nitrogen removal along with desalination and bioelectricity generation.

Use of nitrifying and denitrifying bacteria to remove ammonia from waste water had been studied for a long time due to their high efficiency and low cost. Nitrifying bacteria not only grow slowly but also require high concentration of oxygen to facilitate the nitrifying process. Moreover, the followed denitrifying process needs the supply of adequate carbon sources for denitrifying bacteria to avoid greenhouse gas emission from the system. It shows the operational control to remove ammonia from waste water would be very difficult. Therefore, it is important to study the physiological and biochemical characteristics of those nitrifying and denitrifying bacteria closely. In 1995, Mulder discovered the disappearance of ammonium at the expense of nitrate and nitrogen production from their denitrifying pilot plant in the Netherlands, then van de Graaf verified an ANAMMOX reaction in the laboratory. Further studies that have revealed the combination of aerobic nitrification and anaerobic ammonium oxidation is more efficient to remove ammonia than most conventional methods. The ANAMMOX process is performed by a group of Planctomycete which involves the oxidation of ammonia anaerobically with nitrite as the final electron acceptor to yield gaseous nitrogen. Since this process is no need of supply external carbon source and oxygen, the ANAMMOX system can offer the advantages of less cost, less microbial contamination and less N2O and NO emission to the environment. This study is to summarize the bacterial species diversity, distribution in nature, their physiological characteristics, and potential biochemical pathways of those nitrogen converting microorganisms. In addition, several novel nitrogen removal technologies are also discussed for further understanding of the process optimization under both aerobic and anaerobic conditions.

Anammox (anaerobic ammonium oxidation) process is a one-step conversion of ammonia into nitrogen gas with nitrite as an electron acceptor. It has been developed as a sustainable technology for ammonia removal from wastewater in the last decade. For wastewater treatment, anammox biomass was widely developed as microbial aggregate where the conditions for enrichment of anammox community must be delicately controlled and growth of other bacteria especially NOB should be suppressed to enhance nitrogen removal efficiency. Little is known about the distribution of microbial aggregates in anammox process. Thus the objective of our study was to assess whether segregation of biomass occurs in granular anammox system. In this study, a laboratory-scale sequential batch reactor (SBR) was successfully operated for a period of 80 days with granular anammox biomass. Temporal and spatial distribution of microbial aggregates was studied by particle characterization system and the distribution of functional microbial communities was studied with qPCR and 16s rRNA amplicon pyrosequencing. Our study revealed the spatial and temporal distribution of biomass aggregates based on their sizes and density. Granules (>200 μm) preferentially accumulated in the bottom of the reactor while floccules (30-200 μm) were relatively rich at the top layer. The average density of aggregate was higher at the bottom than the density of those at the top layer. Degranulation caused by lack of hydrodynamic shear force in the top layer was considered responsible for this phenomenon. NOB was relatively rich in the top layer while percentage of anammox population was higher at the bottom, and anammox bacteria population gradually increased over a period of time. NOB growth was supposed to be associated with the increase of floccules based on the concurrent occurrence. Thus, segregation of biomass can be utilized to develop an effective strategy to enrich anammox and wash out NOB by shortening the settling time and withdrawing floccular biomass from the top of the reactor.

There is a significant need to remove ammonia from municipal wastewater to meet increasingly stringent regulations set by Canada, US, and Europe. Although existing conventional biological wastewater treatment technologies are shown to achieve effective ammonia treatment, they are substantially limited by increased operational intensity and cost. Due to these limitations, other cost-effective biological treatment technologies, such as partial nitritation/anammox (PN/A), have become a more attractive solution for nitrogen removal at wastewater resource recovery facilities (WRRF). A moving bed biofilm reactor system (MBBR) operating under a novel design strategy using elevated total ammonia nitrogen (TAN) loading rate has shown promise to achieve robust partial nitritation and the oxidation of TAN with limited oxidation of nitrite without the need for intense operational measures. However, the novel and promising design strategy using elevated TAN loading rate was applied at higher influent TAN concentrations that are typically greater than concentrations in mainstream municipal wastewater. Therefore, the objective of this dissertation is to investigate and optimize the design and performance of a promising elevated loaded partial nitritation MBBR technology for mainstream, municipal wastewater treatment followed by downstream anammox to complete the design of a robust, stable, energy-efficient, and low operational cost total nitrogen removal PN/A system for mainstream wastewaters. The first specific objective of the dissertation is to isolate the optimal design parameter of a mainstream elevated loaded partial nitritation MBBR system. The results identifies optimal distinct elevated surface area loading rate (SALR), hydraulic retention time (HRT), and airflow rate that achieve stable partial nitritation performance (i.e., optimum total ammonia nitrogen (TAN) removal kinetics and percent NOₓ as nitrite) in a mainstream elevated loaded partial nitritation MBBR system. The study shows that TAN SALR, HRT, and airflow rate significantly affect TAN surface area removal rates (SARR) and percent NOₓ as nitrite and, as such, identifies the optimal design parameters (TAN SALR, HRT and airflow rate) of a mainstream elevated loaded partial nitritation MBBR system. A TAN SALR of 5 g TAN/m²∙d, HRT of 2h and airflow rate of 1.5 L/min are identified to provide stable partial nitritation performance with a TAN SARR of 2.3 ± 0.3 g TAN/m²∙d and a percent of NOx as nitrite of 84.8 ± 1.2% in the mainstream elevated loaded partial nitritation MBBR system. The second specific objective further identifies a new design configuration and the mechanism of nitrite oxidation suppression of the mainstream elevated loaded partial nitritation MBBR technology. The results identifies a unique design strategy using an elevated TAN SALR of 5 g TAN/m²∙d to achieve cost-effective, stable, and elevated rates of partial nitritation in an MBBR system under mainstream conditions. The elevated loaded partial nitritation MBBR system achieves a TAN SARR of 2.01 ± 0.1 g TAN/m²∙d and NO₂⁻-N:NH₄⁺-N stoichiometric ratio of 1.15:1, which is appropriate for downstream anammox treatment. The elevated TAN SALR design strategy promotes nitrite-oxidizing bacteria (NOB) activity suppression rather than a reduction in NOB population as the reason for the suppression of nitrite oxidation in the mainstream elevated loaded partial nitritation MBBR system. NOB activity is limited at an elevated TAN SALR, likely due to thick biofilm embedding the NOB population and competition for dissolved oxygen (DO) with ammonia-oxidizing bacteria for TAN oxidation to nitrite within the biofilm structure, which ultimately limits the uptake of DO by NOB in the system. The third specific objective of this research characterizes the effects of distinct mixing and aeration strategies on the performance of the mainstream elevated loaded partial nitritation MBBR technology. This is addressed through a study investigating and comparing the kinetics, biofilm characteristics, and embedded biomass of three distinct mixing and aeration strategies employed to operate the mainstream elevated loaded partial nitritation MBBR system. The study compares the conventional mixing and aeration condition, continuous aeration with mechanical paddle & aeration, and recirculation pump & aeration utilized to optimize the partial nitritation MBBR system to achieve low DO effluent concentrations for optimal downstream anammox treatment. The results show that maintaining mixing and aeration in the elevated loaded partial nitritation MBBR system with recirculation pump & reduced aeration achieves lower effluent DO concentration and stable partial nitritation with appropriate NO₂⁻-N:NH₄⁺-N stoichiometry ratio of 1.09:1 for subsequent anammox treatment compared to operation with continuous aeration or mechanical paddle & aeration. The fourth specific objective of this research investigates the promising elevated loaded PN/A configured system for nitrogen removal under mainstream conditions. This is achieved through the operation of the elevated loaded partial nitritation MBBR system following the anammox unit as a combined two-stage system for nitrogen removal at mainstream municipal concentration. The elevated loaded partial nitritation MBBR system provides optimal NH₄⁺-N:NO₂⁻-N stoichiometric effluent ratio of 1:1.17, resulting in the successful operation of a downstream anammox unit with a total nitrogen removal rate at 0.22 ± 0.2 g N/m²/d and total nitrogen removal efficiency at 74.1 ± 0.7%. The average NO₂⁻-N to NH₄⁺-N molar removal ratio is 1.05 ± 0.1 from the anammox unit. Also, the anammox bacteria (AnAOB) gene copies are at 3.28 ± 0.7 × 10⁸, a value significantly higher than the AOB and NOB gene copies at 9.17 ± 1.1 × 10⁴ and 6.23 ± 1.0, respectively. This confirms that anammox activity is established and nitrogen removal is primarily through the anammox process. The results and overall system performance demonstrate that the combined two-stage mainstream elevated loaded partial nitritation/anammox MBBR system has shown promise and offers great insights for further advancement of the anammox process at mainstream municipal wastewaters. Finally, the economic evaluation and cost comparative analyses conducted show that compared to the conventional biological nitrification/denitrification process for nitrogen removal, the two-stage elevated loaded PN/A system offers a 57.6% savings on energy cost, 100% savings on chemical cost, and 68.7% savings on the cost of sludge disposal. Therefore, the two-stage elevated loaded PN/A system, in addition to high nitrogen removal efficiency, reduced footprint, and ease of operation, is also economically favorable and reduces the overall operational cost of wastewater treatment system by 61.6%, thus saving up to an average of 3.7 million CAD every year.

Anaerobic ammonium oxidizing (Anammox) bacteria are known to utilize ammonium and nitrite as electron donor and acceptor, respectively, to produce nitrogen gas as their main final product with by-product formation of nitrate. Anammox bacteria provide the advantages of significant saving in aeration, no requirement for external electron donor, reduction of greenhouse gas emission, lowered sludge production, and higher specific nitrogen-removing activity compared to the conventional nitrification-denitrification process used in nutritent-N removal. Therefore, the anammox process has recently been widely studied and applied as a state-of-the-art biotechnology to remove nutrient nitrogen from ammonium-rich wastewater. However, the inhibitory impact of nitrite (one of the two main substrates) on the anammox process has been reported in both lab- and full-scale anammox systems, which limits the application of anammox process. Based on the current knowledge, a wide range of nitrite concentrations causing anammmox inhibition was reported to be correlated to the pH and energy status of anammox bacteria, and the understanding of the mechanisms of nitrite inhibition to anammox bacteria is still not clear. Therefore, the purpose of this work is to investigate the mechanism of nitrite inhibition and develop a strategy for recovering nitrite inhibited anammox processes. The effects of pre-exposing anammox bacteria to nitrite alone on their subsequent activity and metabolism after ammonium has been added was evaluated in batch bioassays. The results showed that pre-exposure of anammox bacteria to nitrite without ammonium caused dramatic inhibition with observed 50% inhibition concentration (IC₅₀) of 52 mg NO₂⁻-N L⁻¹, compared to an IC₅₀ of 384 mg NO₂⁻-N L⁻¹ obtained in the control group with ammonium and nitrite added simultaneously. The accumulated nitric oxide (NO) found in the group with anammox bacteria pre-inhibited by nitrite indicated that pre-exposure to nitrite most likely caused disruption of the anammox biochemistry by interrupting the hydrazine synthesis step. Meanwhile, active metabolic status of anammox bacteria fueled by a strong proton gradient maintained by controlling pH in the optimal range of 7.2-7.8 enhanced the ability of anammox bacteria to tolerate nitrite inhibition. This was evaluated by depleting the proton gradient by utilizing two uncouplers of respiration, 2,4 dinitrophenol (24DNP) and carbonyl cyanide m-chlorophenyl hydrazine (CCCP). The results showed that presence of 0.28 mg CCCP L⁻¹ caused enhancement of nitrite inhibition to anammox bacteria, with a calculated IC₅₀ of 18.7 mg NO₂⁻-N L⁻¹ compared to an IC₅₀ greater than 150 mg NO₂⁻-N L⁻¹ in the control group lacking CCCP. Meanwhile, the sensitivity to NO₂⁻ was 3 times in anammox bacteria pre-exposed to 100 mg NO₂⁻ L⁻¹ for 24 h than in treatments lacking 37.8 mg 24DNP L⁻¹. A potential strategy of detoxifying the nitrite inhibition to anammox bacteria was proposed by using nitrate due to the finding of the presence of NarK, with potential function of NO₃⁻/NO₂⁻ antiporter, encoded in the anammox genome. Both batch- and continuous-experiments were carried out to test this hypothesis. The relative contribution of nitrate to nitrite detoxification was found to be pH dependent but the attenuation of nitrite inhibition is independent of the proton motive force which is supported by the result that nitrate caused almost complete attenuation of nitrite toxicity in cells exposed to the proton gradient disruptor, CCCP, at pH 7.5. Increase in nitrate concentration also improved the attenuation of nitrite inhibition to anammox process, with the maximum recovery being achieved at 0.85 mM in batch experiment and 2.0 mM for 3 days in continuous-fed bioreactor. Moreover, the timing of nitrate addition is significant because long-term nitrite inhibition of anammox biomass results in irreversible damage of the cells, under which condition addition of nitrate showed no positive impact on recovery of nitrite inhibition. This study also investigated the inhibitory effects of six metals (Cu²⁺, Cd²⁺, Ni²⁺, Zn²⁺, Pb²⁺, and molybdate) commonly found in landfill leachate on anammox activity. Results from batch bioassays indicated that precipitation reactions decreased considerably the soluble concentration of the cationic metals. Cu, Zn, Cd, and Ni were the most toxic metals with 50% inhibiting soluble concentrations of 4.2, 7.6, 11.2, and 48.6 mg L⁻¹, respectively. Molybdate and Pb²⁺ were not or only moderately inhibitory at the highest soluble concentrations tested (22.7 mg Mo L-1 and 6.0 mg Pb L⁻¹, respectively). Microbial inhibition was strongly correlated with both the added- and the dissolved metal concentration. These relationships could be described by a noncompetitive inhibition model for all inhibitory metals except for Pb. The results of this dissertation indicate that the resistance of anammox bacteria to nitrite inhibition could be enhanced by maintaining either an active metabolism in simultaneous presence of ammonium and nitrite, or sufficient proton gradient to enable relieving nitrite accumulation in sensitive regions of the anammox cells through an active nitrite transport system. An alternative nitrite detoxification mechanism was also demonstrated which relied on a secondary transport system facilitated by exogenous nitrate to avoid the accumulation of toxic intraorganelle nitrite concentration. Moreover, the results obtained in the study investigating the impact of heavy metals on anammox process provides new insights on the sensitivity of anammox bacteria to common metals and can be used to devise strategies to minimize inhibition of the anammox process when treating wastewater containing heavy metals.

The seasonal variation of anammox is yet to be comprehensively studied, unlike denitrification, the more traditional sink for fixed nitrogen. A seasonal study of anammox, denitrification and benthic oxygen consumption using the revised isotope pairing technique is presented in Chapter 2. Experimental temperature and NO3- concentration were kept constant throughout so that the capacity of the sediment for anammox could be estimated. Similar seasonal variations in the rates of anammox, denitrification and oxygen consumption suggest that anammox is controlled by the availability of organic carbon. Furthermore the effect of tidal inundation by overlying water rich in NO3- was investigated by measuring rates of anammox, denitrification and oxygen consumption at three tidal elevations throughout the year. A significant relationship between anammox and denitrification was established at each tidal elevation, which increased in strength as length of inundation decreased. To complement this seasonal study, additional experiments were undertaken, which are described in Chapter 3, to determine how anammox, denitrification and sediment metabolism responds to variations in experimental NO3- concentration and temperature. There were significant increases in rates of anammox, denitrification and sediment metabolism with temperature until 20oC when rates of anammox began to reduce. Furthermore there was significant variation in the response of all three processes to temperature in samples collected at different dates, which suggested that reduced bioavailability of organic carbon in the winter months was limiting the response to temperature. In addition to exploring how inorganic N is cycled in estuarine sediments, the ability of estuarine sediments to oxidize urea via nitrite was examined using 15N and 13C labelled substrates. Results, which are presented in Chapter 4, indicate that urea added to anaerobic sediment slurries was rapidly hydrolysed to ammonium before being oxidized via the anammox pathway.

The anaerobic oxidation of ammonium (anammox) with nitrite as electron acceptor is a microbial process that generates nitrogen gas as main final product. After being discovered in the Netherlands in the 1990s, anammox has been applied in state-of-the-art biotechnologies for the removal of N pollution from ammonium rich wastewaters. The anammox process offers significant advantages over traditional nitrification-denitrification based processes. Since anammox does not need elemental oxygen, it allows for important savings in aeration. Furthermore, due to the autotrophic nature of the bacteria, anammox does not require external addition of electron donor, often needed in systems with post-denitrification. Although the anammox bacteria have high specific activity, they are slow growing, with doubling times that can range from 10 to 25 d. Therefore, in case of a toxic event causing the death of the biomass, a long recovery period will be required to reestablish full treatment capacity. The purpose of this work is to investigate the inhibition of anammox bacteria by compounds commonly found in wastewaters, including substrates, intermediates and products of the anammox reaction. Among common wastewater constituents, sulfide was shown to be especially harmful, causing complete inhibition of anammox activity at concentrations as low as 11 mg H₂S L⁻¹. Dissolved oxygen was moderately toxic with a 50% inhibiting concentration of 2.3 and 3.8 mg L⁻¹ to granular and suspended anammox cultures, respectively. Among the various compounds involved in the anammox reaction, special attention was paid to nitrite. Numerous literature reports have indicated inhibition of anammox bacteria by its terminal electron acceptor. However to date, there is no consensus explanation as to the mechanism of nitrite inhibition nor on how the inhibition is impacted by variations in the physiological status of anammox cells. The mechanisms of anammox inhibition by nitrite were thoroughly investigated in batch and continuous experiments of this dissertation. The results of this work demonstrate that conditions hindering generation of metabolic energy have a detrimental effect on the tolerance of anammox cells to toxic levels of nitrite. The absence of ammonium during events of nitrite exposure was shown to exacerbate its toxic effect. As a result of nitrite inhibition, nitric oxide, an intermediate of the anammox reaction, accumulated in the head space of the batch experiments. Moreover, nitrite inhibition was enhanced at the lowest range of pH tested (6.4-7.2), while same nitrite concentrations caused no inhibition under mildly alkaline conditions (7.5-7.8). Although other authors have relied on the classic concept that undissociated nitrous acid is the species responsible for the inhibition, the results in this work indicate that the pH affects the inhibitory effect of nitrite, irrespective of the free nitrous acid concentration. Nitrite stress triggered an active response of the anammox bacteria, which temporarily increased their ATP content to mitigate the inhibition. Additionally, starvation of anammox microorganisms, caused during storage or by sustained underloading of bioreactors, was found to limit the capacity of the bacteria to tolerate exposure to nitrite. The results of this dissertation indicate that the tolerance of anammox bacteria to NO₂⁻ inhibition relies on limiting its accumulation in sensitive regions of the cell. Active metabolism in presence of NH₄⁺ allows for active consumption of NO₂⁻, avoiding accumulation of toxic intracellular NO₂⁻ concentrations. Furthermore, secondary active transport proteins may be used by anammox bacteria to translocate nitrite to non-sensitive compartments. Nitrite active transport relies on a proton motive force. Therefore, conditions such as low pH (below 7.4) or absence of energy sources, which may disturb the maintenance of the intracellular proton gradient, will increase the sensitivity of anammox cells to NO₂⁻ inhibition. Strategies for the operation and control of anammox bioreactors must be designed to avoid exposure of the biomass to nitrite under the absence of ammonium, low pH or after periods of starvation.

In today’s progressively urbanised society, there is an increasing need for cost-effective, environmentally sound technologies for the removal of nutrients (carbon, phosphorous, nitrogen) from polluted water. Nitrogen removal from wastewater is the focus of this thesis. Conventional nitrogen removal requires the two processes of aerobic nitrification followed by anoxic denitrification, which is driven by remaining reducing power. While most wastewaters contain a significant fraction of reducing power in the form of organic substrate, it is difficult to preserve the reducing power required for denitrification, due to the necessary preceding aerobic oxidation step. Consequently, one of the major limitations to complete N-removal in traditional wastewater treatment systems is the shortage of organic carbon substrate for the reduction of oxidised nitrogen (NO2-, NO3-), produced from nitrification. This thesis followed two main research themes that aimed to address the problem of organic carbon limitation in nitrogen removal from wastewater, by management of the oxygen supply. The first theme was the study of N-removal by simultaneous nitrification and denitrification (SND) in the novel reactor type, the sequencing batch reactor (SBR). It was aimed to increase understanding of PHB metabolism and the limiting factors of SND and then to develop a suitable on-line control strategy to manage the oxygen supply and optimise nitrogen removal by SND. The second main research theme was the application of the CANON(Completely Autotrophic Nitrogen-removal Over Nitrite) process for nitrogen removal from wastewater; a novel process that requires minimal oxygen supply and has the potential to completely circumvent the requirement for organic substrate in nitrogen removal because it is catalysed by autotrophic microorganisms – Anammox (anaerobic ammonium oxidisers) and aerobic nitrifiers. For study of the SND process, a completely automated 2 L sequencing batch reactor was developed with on-line monitoring of the dissolved oxygen concentration, pH and oxidation-reduction (ORP) potential. The SBR was operated continuously for up to 2 years and, due to its separation of different phases by time, enabled the study and optimisation of different microbial activities, including acetate uptake and conversion to PHB (feast phase), PHB hydrolysis and consumption (famine phase), nitrification and denitrification (and SND). All experimental work was performed using a mixed culture Project summary and acetate as the organic substrate. Acetate consumption and PHB production was studied under different oxygen supply rates to establish conditions that allow maximum conversion of acetate to PHB during the feast phase. Lower DO supply rates (kLa 6 – 16 h-1) resulted in preservation of a higher proportion of acetate as PHB than at higher DO supply rates (kLa 30 and 51 h-1). Up to 77 % of the reducing equivalents available from acetate were converted to PHB under O2-limitation, as opposed to only 54 % under O2-excess conditions, where a higher fraction of acetate was used for biomass growth. A metabolic model based on biochemical stoichiometry was developed that could reproduce the trends of the effect of oxygen on PHB production. Experimental findings and simulated results highlighted the importance of oxygen control during the feast phase of an SBR in preserving reducing power as PHB. To develop an oxygen management strategy for the aerobic famine phase,the effect of the dissolved oxygen (DO) concentration on SND, using PHB as the electron donor, was investigated. There was a clear compromise between the rate and the percentage of SND achieved at different DO concentrations. A DO setpoint of 1 mg L-1 was optimal for both the percentage of SND (61 %) and rate of SND (4.4 mmol N. Cmol X-1. h-1). Electron flux analysis showed that most SND activity occurred during the first hour of the aerobic famine period, when the oxygen uptake rate (due to NH4 + and PHB oxidation) was highest. Aerated denitrification ceased as soon as NH4 + was depleted. The presence of NH4 + provided an oxygen “shield”, preventing excessive penetration of oxygen into the flocs and creating larger anoxic zones for SND. PHB degradation was first order with respect to the biomass PHB concentration (dfPHB/dt = 0.19 . fPHB). The slow nature of PHB degradation made it a suitable substrate for SND, as it was degraded at a similar rate to ammonium oxidation. While DO control during the aerobic famine phase could increase nitrogen removal via SND, total N-removal in the SBR was still limited by the availability of reducing power(PHB) in the anoxic phase. The length of the aerobic phase needed to be minimised to prevent over-oxidation of PHB after NH4 + depletion. The specific oxygen uptake rate (SOUR) was found to be an effective on-line parameter that could reproducibly detect the end-point of nitrification. A simple method was developed for continuous, on-line measurement of the SOUR, which was used for automated adjustment of the aerobic phase length. Minimisation of the aerobic phase length by feedback control of the Project summary SOUR improved nitrogen removal from 69 % (without phase length control) to 86 %, during one cycle. The SOUR-aeration control technique could successfully adapt the aerobic phase length to varying wastewater types and strengths and to varying aeration conditions. The medium- and long-term effects of oxygen management on nitrogen removal was investigated by operating the SBR continuously for up to one month using DO control throughout all stages of the SBR, i.e. oxygen-limitation during the feast phase, a DO setpoint of 1 mg L-1 during the famine phase and SOUR controlled aerobic phase length. Complete oxygen management resulted in minimisation of the amount of PHB that was oxidised aerobically in each SBR cycle and caused an accumulation of cellular PHB over time. The increased availability of PHB during aeration resulted in a higher SOUR and increased N-removal by SND from 34 to 54 %. After one month of continuous SBR operation, the settling efficiency of the biomass improved from 110 mL . g-1X to less than 70 mL . g-1X and almost complete N-removal (9 %) was achieved via SND during aeration, however at a reduced rate (1.5 mmol Cmol X-1 h-1). Therefore, long-term oxygen management resulted in biomass with improved settling characteristics and a higher capacity for SND. Results of the first main research theme highlighted the importance of aeration control throughout all stages of the SBR for maximum N-removal via SND. The CANON process was investigated as an alternative to the use of conventional activated sludge for treatment of wastewaters limited by organic carbon substrate. The initial study of the CANON process was performed at the Kluyver Laboratory in Delft, the Netherlands, using an already established Anammox enrichment culture. The effect of extended periods of NH4 +-limitation on the CANON microbial populations was studied, to examine their ability to recover from major disturbances in feed composition. The CANON process was stable for long periods of time until the N-loading rate reached below 0.1 kg N m 3 day-1, when a third population of bacteria developed in the system (aerobic nitrite oxidisers), resulting in a decrease in N-removal from 92 % to 57 %. Nitrite oxidisers developed due to increased levels of oxygen and nitrite. This highlighted the requirement for oxygen control during the CANON process to prevent increased DO levels and growth of undesired microbes. To initiate the CANON process from a local source, Anammox was enriched from local activated sludge (Perth, Western Australia). FISH analysis (fluorescence in situ hybridisation) of the enriched Anammox strain showed that it belonged to the Order Planctomycetales, Project summary the same as all other identified Anammox strains, but represented a new species of Anammox. The enrichment culture was not inhibited by repeated exposure to oxygen, allowing initiation of an intermittently-aerated CANON process to achieve sustained, completely autotrophic ammonium removal (0.08 kg N m-3 day-1) for an extended period of time, without any addition of organic carbon substrate. Dissolved oxygen control played a critical role in achieving alternating aerobic and anaerobic ammonium oxidation. The main conclusion drawn from the study is the important role of oxygen management in achieving improved nitrogen removal. A careful oxygen management strategy can minimise wastage of reducing power to improve PHB-driven SND by activated sludge and can prevent major disturbances to the population balance in the CANON system. Oxygen management has the potential to reduce aeration costs while significantly improving nitrogen removal from wastewaters limited by organic carbon.

Partial denitrification/anammox (PdNA) is an emerging biological nutrient removal (BNR) process that can be used to remove ammonia (NH3) and NOx from wastewater. This process is a combination of partial denitrification (PdN), which serves to reduce nitrate (NO3) to nitrite (NO2), and anaerobic ammonia oxidation, or anammox (AMX), which uses the nitrite as an electron acceptor to oxidize ammonia. PdNA provides significant aeration and external carbon savings when compared to the conventional nitrification/denitrification biological removal process for nitrogen but has been difficult to implement in mainstream treatment conditions due to many factors. One of these factors is the slow growth rate and startup time of anammox bacteria. This research first focused on determining the required startup time and startup optimization methods for a proposed mainstream polishing PdNA MBBR at Hampton Roads Sanitation District's James River Treatment Plant (JRTP). These two MBBRs were started with either virgin carriers or carriers coated with a preliminary biofilm and were fed secondary effluent The MBBRs were dosed with glycerol based on a feedforward carbon control approach and were not seeded with anammox at any point. Anammox activity was detected in the preliminary biofilm and virgin media MBBRs approximately 52 and 86 days after startup, respectively. Based on these results, starting up a mainstream PdNA reactor without seed is possible, and using preliminary biofilm carriers can speed up startup by approximately one month. A second experiment was conducted to determine the carbon demand and nitrogen removal capabilities of a glycerol fed PdNA MBBR and AMX MBBR in series. A nitrifying MBBR was also added to the MBBR train to test how well residual nitrite leaving the MBBRs could be polished off to limit ozone/disinfectant demand downstream. Additionally, a methanol-fed PdNA integrated fixed-film activated sludge (IFAS) reactor was also operated to determine the carbon demand and nitrogen removal capabilities for a PdNA process in an IFAS reactor. The PdNA and AMX MBBRs had average effluent TIN concentrations of 3.75 ± 1.25 and 2.81 ± 1.21 mg TIN/L, respectively, with a COD dosed per TIN removed ratio (COD/TIN) of 2.42 ± 0.77 g COD/g TIN for the entire process. The PdNA IFAS reactor had average effluent TIN concentrations of 4.07 ± 1.66 mg/L and 3.30 ± 0.96 mg/L at hydraulic retention times (HRTs) of 30 and 25 minutes. At these two HRTs, the PdNA IFAS process had a COD/TIN ratio of 1.08 ± 0.38 and 2.18 ± 0.99 g COD/g TIN, respectively. Overall, this indicated that both the PdNA MBBR and IFAS processes could reach low effluent TIN limits in mainstream conditions with low demand for COD, even with relatively low and unstable PdN efficiencies. Additionally, the nitrifying MBBR managed to keep the effluent nitrite concentration consistently below 0.5 mg/L at ammonia and nitrite influent loadings rates of 0.055 ± 0.035 and 0.379 ± 0.112 g N/m2/day. This research demonstrated that starting a PdNA process in mainstream conditions, without seed, can be accomplished within a reasonable timeframe and provides knowledge that can help engineers better understand the advantages of PdNA and design and startup mainstream polishing PdNA MBBRs and IFAS reactors. / Master of Science / As the human population continues to grow and wastewater discharge requirements continue to become more stringent, researchers and engineers have been exploring new technologies and methods to treat wastewater more efficiently. Once such method that is currently being explored is the integration of anaerobic ammonia oxidation, or anammox (AMX), bacteria with a variety of wastewater treatment technologies to remove nitrogen more efficiently from wastewater. AMX synchronously remove ammonia, which exists naturally in wastewater, and nitrite through an oxidation/reduction reaction in which the nitrogen leaves the wastewater in the form of dinitrogen gas. This process greatly reduces the amount of aeration and external carbon needed for the removal of nitrogen from wastewater compared to the commonly used method of full nitrification and denitrification, which are large operational costs at a wastewater treatment plant. While AMX have found use at full-scale plants in treating concentrated sidestreams with the use of partial nitrification (PN) to produce nitrite for the AMX, little progress has been made to integrate AMX into a full-scale mainstream treatment process where the stream is less concentrated and not ideal for consistent PN. Partial denitrification (PdN), however, has shown some promise in reliably producing nitrite in mainstream conditions for AMX usage. On top of the demand for nitrite, AMX bacteria also grow very slowly compared to most bacteria, which means these processes require relatively large amounts of time to get started. A common strategy for decreasing the startup time of AMX processes has been the addition of AMX biomass to a reactor during startup, but this is not feasible in a full-scale mainstream process due to the large amount of biomass that would be required. Therefore, other methods for startup optimization must be evaluated, which this study sought to do through two startup experiments in separate mainstream polishing moving bed biofilm reactors (MBBRs), which use plastic carriers to develop biofilms of bacteria. These two MBBRs were started with different types of carriers in them, one with carriers coated with a pre-established preliminary biofilm and one with brand-new, virgin carriers, to see what kind of effect these different types of carriers have on AMX startup time. AMX activity was detected in the preliminary biofilm and virgin media MBBRs approximately 52 and 86 days after startup, respectively, which was much quicker than expected. This indicates that starting up a mainstream PdNA reactor without seed is possible and using the preliminary biofilm carriers can speed up startup by approximately one month. After the startup experiment, one of the MBBRs was converted to a PdNA integrated fixed-film activated sludge (IFAS) reactor through the addition of activated sludge. This PdNA IFAS reactor was operated alongside a PdNA MBBR and AMX MBBR to test their nitrogen removal and carbon savings capabilities. Operation of these reactors demonstrated that both a PdNA MBBR or IFAS process are capable of consistently removing nitrogen to low levels with relatively low amounts of external carbon addition, even with inconsistent PdN. Overall, this research provided valuable insight into startup methods and design requirements of PdNA MBBRs and IFAS reactors which will make the implementation of these treatment processes more feasible.