Hydroxylamine is a key intermediate in several biological reactions of the global nitrogen cycle. However, the role of hydroxylamine in anammox is still not fully understood. In this work, the impact of hydroxylamine (also in combination with other substrates) on the metabolism of a planktonic enrichment culture of the anammox species Ca. Kuenenia stuttgartiensis was studied. Anammox bacteria were observed to produce ammonium both from hydroxylamine and hydrazine, and hydroxylamine was consumed simultaneously with nitrite. Hydrazine accumulation - signature for the presence of anammox bacteria - strongly depended on the available substrates, being higher with ammonium and lower with nitrite. Furthermore, the results presented here indicate that hydrazine accumulation is not the result of the inhibition of hydrazine dehydrogenase, as commonly assumed, but the product of hydroxylamine disproportionation. All kinetic parameters for the identified reactions were estimated by mathematical modelling. Moreover, the simultaneous consumption and growth on ammonium, nitrite and hydroxylamine of anammox bacteria was demonstrated, this was accompanied by a reduction in the nitrate production. Ultimately, this study advances the fundamental understanding of the metabolic versatility of anammox bacteria, and highlights the potential role played by metabolic intermediates (i.e. hydroxylamine, hydrazine) in shaping natural and engineered microbial communities.

A model-based study was developed to analyse the behaviour of Moving Bed Biofilm Reactor (MBBR) and Integrated Fixed-Film Activated Sludge (IFAS) reactor configurations for the removal of nitrogen in the main water line of municipal wastewater treatment plants via partial nitritation/anammox (PN/AMX). The basic principles and underlying mechanisms linking operating conditions to process performance were investigated, with particular focus on nitrite oxidizing bacteria (NOB) repression and resulting volumetric conversion rates. The external mass transfer resistance is a major factor differentiating granular sludge PN/AMX processes from MBBR or IFAS systems. The external mass transfer resistance was found to promote the metabolic coupling between anammox (AMX) and ammonia oxidizing bacteria (AOB), crucial for NOB repression in the biofilm. Operation at low bulk DO prevents NOB proliferation in the flocs of IFAS systems as AMX activity limits nitrite availability (the so-called AMX nitrite sink). Importantly, the effectiveness of the AMX nitrite sink strongly depends on the AMX sensitivity to oxygen. Also, over a broad range of operational conditions, the seeding of AOB from the biofilm played a crucial role in maintaining their activity in the flocs. From a practical perspective, while low DO promotes NOB repression, lower nitrogen loads have to be applied to maintain the same effluent quality. Thus, a trade-off between NOB repression and volumetric conversion capacity needs to be defined. To this end, IFAS allow for higher volumetric rates, but the window of operating conditions with effective NOB repression is smaller than that for MBBR. Ultimately, this study identified the principles controlling NOB in MBBR and IFAS systems and the key differences with granular reactors, allowing for the interpretation of (seemingly contradictory) published experimental results.

A lab-scale partial nitritation granular sludge air-lift reactor was operated in continuous mode treating low strength synthetic medium (influent ca. 50 mg-N-NH₄ ⁺/L). Granules were initially stratified with AOB in the external shell and NOB in the inner core at 20 °C. Once temperature was decreased progressively from 20 °C to 15 °C, nitrate production was initially observed during several weeks. However, by maintaining relatively high ammonium concentrations in the liquid (ca. 28 mg-N-NH₄ ⁺/L), effluent nitrate concentrations in the reactor decreased in time and process performance was recovered. Batch tests were performed in the reactor at different conditions. To understand the experimental results an existing one-dimensional biofilm model was used to simulate batch tests and theoretically assess the impact of stratification, dissolved oxygen (DO) and short-term effects of temperature on time course concentrations of ammonium, nitrite and nitrate. This theoretical assessment served to develop an experimental methodology for the evaluation of in-situ batch tests in the partial nitritation reactor. These batch tests proved to be a powerful tool to easily monitor the extent of stratification of nitrifier guilds in granular sludge and to determine the required bulk ammonium concentration to minimize nitrite oxidation. When nitrifier guilds were stratified in the granular sludge, a higher bulk ammonium concentration was required to efficiently repress NOB at lower temperature (ca. 19 versus 7 mg-N-NH₄ ⁺/L at 15 and 20 °C, respectively).

A new type of structural extracellular polymers (EPS) was extracted from aerobic granular sludge dominated by ammonium-oxidizing bacteria. It was analyzed by Raman and FTIR spectroscopy to characterize specific amino acids and protein secondary structure, and by SDS-PAGE with different stains to identify different glycoconjugates. Its intrinsic fluorescence was captured to visualize the location of the extracted EPS in the nitrifying granules, and its hydrogel-forming property was studied by rheometry. The extracted EPS is abundant with cross ß-sheet secondary structure, contains glycosylated proteins/polypeptides, and rich in tryptophan. It forms hydrogel with high mechanical strength. The extraction and discovery of glycosylated proteins and/or amyloids further shows that conventionally used extraction and characterization techniques are not adequate for the study of structural extracellular polymers in biofilms and/or granular sludge. Confirming amyloids secondary structure in such a complex sample is challengeable due to the possibility of amyloids glycosylation and self-assembly. A new definition of extracellular polymers components which includes glycosylated proteins and a better approach to studying them is required to stimulate biofilm research.

Cellulose represents a significant fraction of domestic wastewater, but it is not considered as a separate state variable in the conventional Activated Sludge Models (ASM). Cellulose is a very slowly degradable substrate that in traditional wastewater characterisation methods would be characterised partly as slowly biodegradable and partly as inert material. This can be problematic when the same model is used under different temperature conditions or different solid retention times. Also with the emerging attention for cellulose recovery, inclusion of this compound in models helps to assess the impact of cellulose recovery on operations and on operational costs. But also in membrane bioreactors, sieves are used in order to remove fibrous material, mainly cellulose. In this study a modification of ASM1 is proposed, where cellulose is introduced as a separate state variable and it is supposed to degrade with a first order hydrolysis rate. With the aid of this model, the effect of fine sieves is simulated using two alternatives, by either sieving the influent or the activated sludge. The model proved to be useful for operational purposes, and illustrates the need for including cellulose as separate state variable.

In the race to achieve a sustainable urban wastewater treatment plant, not only the energy requirements have to be considered but also the environmental impact of the facility. Thus, nitrous oxide (N₂O) emissions are a key-factor to pay attention to, since they can dominate the total greenhouse gases emissions from biological wastewater treatment. In this study, N₂O production factors were calculated during the operation of a granular sludge airlift reactor performing partial nitritation treating a low-strength synthetic influent, and furthermore, the effect of temperature on N₂O production was assessed. Average gas emission relative to conversion of ammonium was 1.5 ± 0.3% and 3.7 ± 0.5% while the effluent contained 0.5 ± 0.1% and 0.7 ± 0.1% (% N-oxidized) at 10 and 20 °C, respectively. Hence, temperature increase resulted in higher N₂O production. The reasons why high temperature favoured N₂O production remained unclear, but different theoretical hypotheses were suggested.

Highly-loaded aerobic chemical oxygen demand (COD) removal reactors (also known as A-stage) include two main processes, COD removal by heterotrophic biomass and adsorption of COD on the activated sludge. A simple model to describe highly-loaded aerobic COD removal reactors has been developed. A one-year-set of measured data from a full scale wastewater treatment plant has been used to calibrate the efficiency of the adsorption and to evaluate the ability of the mathematical model to describe the measured data in both steady state and dynamic conditions. The approach lumped the efficiency of the settler and the adsorption with a simple but powerful approach which includes the use of the measured sludge retention time (SRT) and the settling efficiency. The effects of dynamics in terms of (i) seasonality (for water temperature, flow rate and concentration of pollutants), and (ii) daily variations in flow rate were investigated. Results showed how during winter the low water temperatures negatively impacted the efficiency of the A-stage, producing a higher COD concentration in the effluent, which eventually could impact the performance of the nitrogen removal in the B-stage. General guidelines for the application of the model to similar highly loaded A-stage reactor systems were provided.

Sewage treatment with anammox could be implemented through a two-step reactor system, where the first reactor would be devoted to partial nitritation. A process design was sketched including control loops. The control strategy regulates the flow-rate of the rich ammonium sidestream produced after dewatering the digested sludge, to keep the ammonium concentration at a set point in the partial nitritation reactor by DOsing the SIde Stream (DOSIS). A second control loop manages the ammonium concentration set point based on the measurement of the total nitrogen in the partial nitritation reactor. A mathematical model was developed to assess the amount of sidestream required. Even in the case of a strong diurnal variability, simulations show how the control strategy is correctly performing, demonstrating the potential of the proposed technology.

This model-based study investigated the mechanisms and operational window for efficient repression of nitrite oxidizing bacteria (NOB) in an autotrophic nitrogen removal process. The operation of a continuous single-stage granular sludge process was simulated for nitrogen removal from pretreated sewage at 10°C. The effects of the residual ammonium concentration were explicitly analyzed with the model. Competition for oxygen between ammonia-oxidizing bacteria (AOB) and NOB was found to be essential for NOB repression even when the suppression of nitrite oxidation is assisted by nitrite reduction by anammox (AMX). The nitrite half-saturation coefficient of NOB and AMX proved non-sensitive for the model output. The maximum specific growth rate of AMX bacteria proved a sensitive process parameter, because higher rates would provide a competitive advantage for AMX.