Currently, the most cost-effective and efficient method for phosphorus (P) removal from wastewater is enhanced biological P removal (EPBR) via polyphosphate-accumulating organisms (PAOs). This study integrates a literature review with genomic analysis to uncover the phylogenetic and metabolic diversity of the relevant PAOs for wastewater treatment. The findings highlight significant differences in the metabolic capabilities of PAOs relevant to wastewater treatment. Notably, Candidatus Dechloromonas and Candidatus Accumulibacter can synthesize polyhydroxyalkanoates, possess specific enzymes for ATP production from polyphosphate, and have electrochemical transporters for acetate and C4-dicarboxylates. In contrast, Tetrasphaera, Candidatus Phosphoribacter, Knoellia, and Phycicoccus possess PolyP-glucokinase and electrochemical transporters for sugars/amino acids. Additionally, this review explores various detection methods for polyphosphate and PAOs in activated sludge wastewater treatment plants. Notably, FISH-Raman spectroscopy emerges as one of the most advanced detection techniques. Overall, this review provides critical insights into PAO research, underscoring the need for enhanced strategies in biological phosphorus removal.

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Kaumera are extracellular polymeric substances (EPS) extracted from excess aerobic granular sludge from Nereda® wastewater treatment plants. Kaumera exhibits significant market potential across diverse applications, fostering rapid research and business development. Furthermore, it will begin to be extracted from numerous installations worldwide. This calls for standard methods as analogue to (waste)water and sludge characterization. Due to lack of standardization, stakeholders are currently using different extraction and characterization protocols, impeding the development of a more uniform product and comparison of results across research studies. To address this, this report compiles the standard protocol for Kaumera extraction in the laboratory and for on-site and lab characterization to be used by researchers, the public Dutch water authorities, and the private industry. The procedures detailed in this document are in accordance with EPS research conducted at TU Delft and methodologies employed in Kaumera production facilities. This report aids in monitoring Kaumera characteristics worldwide and for optimizing the extraction process (including up and downstream processing). This will help maximize repeatability, interoperability, and quality and therefore accelerate business and research development, paving the way to develop a product that meets the needs of the endusers. Through the widespread adoption of this manual, our aim is to foster greater coordination and collaboration among stakeholders, thereby expediting the realization of Kaumera's full potential. @en

Aerobic granular sludge (AGS) is an innovative biotechnology extensively applied for treating municipal wastewater, and it can potentially treat sugar industry wastewater. Glucose is a prevalent substrate in sugar industry wastewater; nevertheless, the effect of glucose on AGS systems remains unexplored. In this study, an AGS reactor using glucose as the sole carbon source was operated in anaerobic-aerobic cycles. The system maintained a solids retention time (SRT) of 10 days, resulting in good granulation and enhanced biological phosphorus removal (EBPR) performance. The glucose fed was rapidly taken up within 10 minutes, with a portion stored as intracellular polymers such as glycogen and poly-hydroxy-alkanoates (PHAs), while another portion underwent anaerobic fermentation to lactate and formate. The carbon balance was not completely closed, with 16% of the carbon speculated to be utilized for the production of an unidentified polymer. The microbial community consisted of diverse organisms, with Micropruina identified as the most abundant genera and Ca. Accumulibacter (a typical type of PAOs) as the second most abundant genera based on metagenomic analysis. A batch test was conducted by adding an excess of glucose, lactate, and formate, revealing that lactate was the probable substrate utilized by PAOs. Additionally, Micropruina was hypothesized to be involved in glucose consumption, glycogen storage, and lactate production. Micropruina and Ca. Accumulibacter collaborate in utilizing glucose, providing them with a significant competitive advantage within the system. Due to their slow growth rate, these bacteria play a crucial role in achieving favorable granulation when supplied with glucose. Promoting the growth of these organisms can be a valuable strategy in engineering applications.

Dormancy is a topic that is researched extensively for various microorganisms, especially in the field of medicine. However, not many studies have been conducted for the dormancy of microorganisms in the wastewater treatment sector. Treatment processes like the NEREDA® technology use Phosphate Accumulating Organisms (PAO) to remove phosphate from the wastewater. If dormancy affects these bacteria, it can cause the wastewater treatment plants to function at a lower capacity. Reviving dormant PAOs can increase treatment capacity and treat the wastewater during high influent loading without effluent deterioration. Studying the optimal conditions for granule storage without inducing dormancy can be advantageous during granule transport for inoculation of reactors during their start-up phase. This study presents the first independent assessment of the possible dormancy of PAOs in Aerobic Granular Sludge (AGS).

In this research thesis, dormancy is a survival strategy opted by PAOs to persist substrate limitation. There are two different pathways to dormancy that has been explored here. Responsive switching and starvation are studied as they express fast and slow transition respectively into dormancy. Lag faced during resuscitation of dormant cells is considered proportional to the length of dormancy in responsive switching.

The experimental phase of the research can be interpreted in three parts: first through method development, second through method testing and third through the application of the developed method. The method was developed to understand dormancy in PAOs by observing differences in their maintenance and activity rates. This method was then tested on granules where dormancy was induced by lowering the temperature to 4ºC and anaerobically storing without feed. This method was then applied to newly sampled granules from a full-scale reactor to study dormancy in the PAOs. The developed method increased the activity of the former granules by 60% but did not raise the activity of the latter. Maintenance rates increased by 167% and 68%, respectively, in the granules. Although dormancy could not be sufficiently proved by the method, starvation of PAOs in Utrecht NEREDA® reactors was possible.

High concentrations of particulate COD (pCOD) in the influent of aerobic granular sludge (AGS) systems are often associated to small granule diameter and a large fraction of flocculent sludge. At high particulate concentrations even granule stability and process performance might be compromised. However, pilot-or full-scale studies focusing on the effect of real wastewater partic-ulates on AGS are scarce. This study describes a 3-month period of increased particulate loading at a municipal AGS wastewater treatment plant. The pCOD concentration of the influent increased from 0.5 g COD/L to 1.3 g COD/L, by adding an untreated slaughterhouse wastewater source to the influent. Sludge concentration, waste sludge production and COD and nutrient removal performance were monitored. Furthermore, to investigate how the sludge acclimatises to a higher influent particulate content, lipase and protease hydrolytic activities were studied, as well as the microbial community composition of the sludge. The composition of the granule bed and nutrient removal efficiency did not change considerably by the increased pCOD. Interestingly, the biomass-specific hydrolytic activities of the sludge did not increase during the test period either. However, already during normal operation the aerobic granules and flocs exhibited a hydrolytic potential that ex-ceeded the influent concentrations of proteins and lipids. Microbial community analysis also revealed a high proportion of putative hydrolysing and fermenting organisms in the sludge, both during normal operation and during the test period. The results of this study highlight the robust-ness of the full-scale AGS process, which can bear a substantial increase in the influent pCOD concentration during an extended period.

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Complex substrates, like proteins, carbohydrates, and lipids, are major components of domestic wastewater, and yet their degradation in biofilm-based wastewater treatment technologies, such as aerobic granular sludge (AGS), is not well understood. Hydrolysis is considered the rate-limiting step in the bioconversion of complex substrates, and as such, it will impact the utilization of a large wastewater COD (chemical oxygen demand) fraction by the biofilms or granules. To study the hydrolysis of complex substrates within these types of biomass, this paper investigates the anaerobic activity of major hydrolytic enzymes in the different sludge fractions of a full-scale AGS reactor. Chromogenic substrates were used under fully mixed anaerobic conditions to determine lipase, protease, α-glucosidase, and β-glucosidase activities in large granules (>1 mm in diameter), small granules (0.2–1 mm), flocculent sludge (0.045–0.2 mm), and bulk liquid. Furthermore, composition and hydrolytic activity of influent wastewater samples were determined. Our results showed an overcapacity of the sludge to hydrolyze wastewater soluble and colloidal polymeric substrates. The highest specific hydrolytic activity was associated with the flocculent sludge fraction (1.5–7.5 times that of large and smaller granules), in agreement with its large available surface area. However, the biomass in the full-scale reactor consisted of 84% large granules, making the large granules account for 55–68% of the total hydrolytic activity potential in the reactor. These observations shine a new light on the contribution of large granules to the conversion of polymeric COD and suggest that large granules can hydrolyze a significant amount of this influent fraction. The anaerobic removal of polymeric soluble and colloidal substrates could clarify the stable granule formation that is observed in full-scale installations, even when those are fed with complex wastewaters. Key points: • Large and small granules contain >70% of the hydrolysis potential in an AGS reactor. • Flocculent sludge has high hydrolytic activity but constitutes <10% VS in AGS. • AGS has an overcapacity to hydrolyze complex substrates in domestic wastewater. <!-- Query ID="Q2" Text=" Graphical abstract contains text below the minimum required font size of 6pts inside the artwork, and there is no sufficient space available for the text to be enlarged. Please provide replacement figure file." -->Graphical abstract: [Figure not available: see fulltext.]@en

Aerobic granular sludge is seen as the future standard for industrial and municipal wastewater treatment. Through a Dutch research and development program, a full-scale aerobic granular biomass technology has been developed – the Nereda® technology – which has been implemented to treat municipal and industrial wastewater. The Nereda ® system is considered to be the first aerobic granular sludge technology applied at full-scale and more than 40 municipal and industrial plants are now in operation or under construction worldwide. Further plants are in the planning and design phase, including plants with capacities exceeding 1 million PE. Data from operational plants confirm the system’s advantages with regard to treatment performance, energy-efficiency and cost-effectiveness. In addition, a new possibility for extracting alginate-like exopolysaccharides (ALE) from aerobic granular sludge has emerged which could provide sustainable reuse opportunities. The case is therefore made for a shift away from the ‘activated sludge approach’ towards an ‘aerobic granular approach’, which would assist in addressing the challenges facing the wastewater treatment industry in Asia and beyond.

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Cellulose is a biopolymer commonly used as a renewable constituent in the production of green materials. Wastewater solids can be an attractive source of cellulose, mainly originating from toilet paper. Cellulosic solids can be recovered (i) through sieving before the sewage treatment - resulting in a Fine Sieve Fraction (FSF), or (ii) after biological treatment in the waste sludge – such as in Nereda® Excess Sludge (NES). Its recovery and valorisation into high-end bio-based products, such as crystalline nanocellulose, could improve the techno-economic feasibility of cellulose recovery from wastewater. A cellulose extraction method from wastewater solids was developed during this project. Reference materials (microcrystalline cellulose and toilet paper) and wastewater-extracted cellulosic pulps were studied for the production of cellulose nanocrystals (CNCs). CNCs were prepared by controlled acid hydrolysis followed by centrifugation, filtration, dialysis, neutralisation and ultrasonication. A detailed protocol for CNCs isolation from toilet paper and wastewater-recovered pulps can be found in the appendix section. CNCs could be extracted at around 30 % yield (g g pulp-1). FSF showed to be an attractive source of nanocellulose since the yield per g FSF was 22 %, while for NES it was only 4 %. The wastewater-CNCs presented rod-like morphology with high aspect ratio (10 – 14), crystallinity (62 – 68 %), and chemical structure very similar to commercially available CNCs. However, there is still great extent for improvements since impurities might be still present and the isolation process needs reproducibility and optimization. Bionanocomposites consisting of CNCs (commercial and lab-made) and alginate materials were investigated by measuring the stiffness of freestanding films. A 50 % loading of commercial-CNCs reinforced alginate films by a factor of 1.6 (19 GPa). For the same loading, CNCs isolated from toilet paper and FSF reinforced alginate by a factor of 1.8 and 1.5 (22 – 18 GPa) respectively. Both characterisation results and reinforcement efficiency of CNCs isolated from toilet paper and wastewater-recovered pulps confirm the good quality of this potential product. Furthermore, we have observed changes in the nematic-isotropic phase diagram of CNCs in alginate suspensions. We developed a model hypothesizing that an alginate-shell decorates the CNCs rods increasing its volume fraction and allowing the nematic phase to appear at lower concentrations. The model was highly representative to the experimental data. Lastly, the market relevance of wastewater-CNCs is discussed together with a research outlook.

Aerobic granular sludge is seen as the future standard for industrial and municipal wastewater treatment. Through a Dutch research and development program, a full-scale aerobic granular biomass technology has been developed – the Nereda® technology – which has been implemented to treat municipal and industrial wastewater. The Nereda® system is considered to be the first aerobic granular sludge technology applied at full-scale and more than 30 municipal and industrial plants are now in operation or under construction worldwide. Further plants are in the planning and design phase, including plants with capacities exceeding 1 million PE. Data from operational plants confirm the system’s advantages with regard to treatment performance, energy-efficiency and cost-effectiveness. In addition, a new possibility for extracting alginate-like exopolysaccharides (ALE) from aerobic granular sludge has emerged which could provide sustainable reuse opportunities. The case is therefore made for a shift away from the ‘activated sludge approach’ towards an ‘aerobic granular approach’, which would assist in addressing the challenges facing the wastewater treatment industry in Asia and beyond.@en

Discharging untreated wastewater will contaminate the surface waters and can lead to spread of diseases and long term ecological damage. The most common method for treatment is by the activated sludge process. In this process, nutrients like nitrogen, phosphorus and COD are removed by bacteria grown in flocs. These bacterial flocs are separated from the treated water by settling. Due to the slow settling velocities of these flocs large settling tanks are needed. Settling tanks take up most of the required space for a wastewater treatment plant. Aerobic granular sludge is a compact technology designed to reduce area requirements, save energy while providing excellent effluent quality. Bacteria are grown in granules instead of flocs and have therefore a much higher settling velocity. This reduces the area requirement significantly. So much even so, that external settling tanks are completely omitted. To grow aerobic granules a few selection principles are needed. First, the influent is brought in contact with the biomass in an anaerobic environment. Here COD is converted by the bacteria into storage polymers. These storage polymers are then used for growth in the presence of oxygen hereby removing phosphorus and nitrogen from the bulk liquid. Secondly, a settling pressure is applied by which slow settling biomass is removed from the reactor, thus leading to the formation of granules. In previous research by the PhD students Janneke Beun (principle of aerobic granulation), Merle de Kreuk (basic process technology for granular sludge nutrient removal) and Mari Winkler and Joao Bassin (Microbiology and process engineering aspects of granular sludge) the basic concepts of the granular sludge technology were worked out. In this thesis the effects of several operational conditions on the conversion processes, formation and stability of aerobic granular sludge was studied. The quick implementation of the technology in practice also meant that several important subjects still needed further investigation. To ensure a well-functioning technology in domestic and industrial applications these subjects were studied in more detail (i.e. adsorption, effect of salinity, higher temperature and other substrates). Besides laboratory work also the start-up and performance of one of the first full-scale aerobic granular sludge reactors treating domestic wastewater is described. The ammonium adsorption properties of aerobic granular sludge, activated sludge and anammox granules have been investigated in Chapter 2. During operation of a pilot-scale aerobic granular sludge reactor, a positive relation between the ammonium influent concentration and the ammonium adsorbed was observed. Aerobic granular sludge exhibited much higher adsorption capacity compared to activated sludge and anammox granules. At an ammonium concentration of 30 mg N L-1, adsorption obtained with activated sludge and anammox granules was around 0.2 mg NH4+-N gVSS-1, while aerobic granular sludge from lab- and pilot scale exhibited an adsorption of 1.7 and 0.9 mg NH4+-N gVSS-1, respectively. No difference in the ammonium adsorption was observed in lab-scale reactors operated at different temperatures (20 and 30 ºC). In a lab-scale reactor fed with saline wastewater, we observed that the amount of ammonium adsorbed, decreased considerably when the salt concentration increased. The results indicate that adsorption or better: ion-exchange of ammonium should be incorporated into models for nitrification/denitrification, certainly when aerobic granular sludge is used. Salinity can adversely affect the performance of most biological processes involved in wastewater treatment (Chapter 3). The effect of salt (NaCl) on the main conversion processes in an aerobic granular sludge (AGS) process accomplishing simultaneous organic matter, nitrogen, and phosphate removal was evaluated in this chapter. Hereto an AGS sequencing batch reactor was subjected to different salt concentrations (0.2 to 20 g Cl- L-1). Granular structure was stable throughout the whole experimental period, although granule size decreased and a significant effluent turbidity was observed at the highest salinity tested. A weaker gel structure at higher salt concentrations was hypothesized to be the cause of such turbidity. Ammonium oxidation was not affected at any of the salt concentrations applied. However, nitrite oxidation was severely affected, especially at 20 g Cl- L-1, in which a complete inhibition was observed. Consequently, high nitrite accumulation occurred. Phosphate removal was also found to be inhibited at the highest salt concentration tested. Complementary experiments have shown that a cascade inhibition effect took place: first, the deterioration of nitrite oxidation resulted in high nitrite concentrations and this in turn resulted in a detrimental effect to polyphosphate-accumulating organisms (PAOs). By preventing the occurrence of the nitrification process and therefore avoiding the nitrite accumulation, the effect of salt concentrations on the bio-P removal process was shown to be negligible up to 13 g Cl- L-1. Salt concentrations equal to 20 g Cl- L-1 or higher in absence of nitrite also significantly reduced phosphate removal efficiency in the system. When aerobic granular sludge is applied for industrial wastewater treatment different soluble substrates can be present. For stable granular sludge formation on volatile fatty acids (e.g. acetate), production of storage polymers under anaerobic feeding conditions has been shown to be important. This prevents direct aerobic growth on readily available COD, which is thought to result in unstable granule formation. In Chapter 4 we investigate the impact of acetate, methanol, butanol, propanol, propionaldehyde and valeraldehyde on granular sludge formation at 35 °C. Methanogenic archaea, growing on methanol, were present in the aerobic granular sludge system. Methanol was completely converted to methane and carbon dioxide by the methanogenic archaeum Methanomethylovorans uponensis during the one-hour anaerobic feeding period, despite the relative high dissolved oxygen concentration (3.5 mg O2 L-1) during the subsequent two-hour aeration period. Propionaldehyde and valeraldehyde were fully disproportionated anaerobically into their corresponding carboxylic acids and alcohols. The organic acids produced were converted to storage polymers, while the alcohols (produced and from influent) were absorbed onto the granular sludge matrix and converted aerobically. Our observations show that easy biodegradable substrates not converted anaerobically into storage polymers could lead to unstable granular sludge formation. However, when the easy biodegradable COD is absorbed in the granules and/or when the substrate is converted by relatively slow growing bacteria in the aerobic period stable granulation can occur. The influence of sludge age on granular sludge formation and microbial population dynamics in a methanol- and acetate-fed aerobic granular sludge system operated at 35 °C is investigated in Chapter 5. During anaerobic feeding of the reactor, methanol was initially converted to methane by methylotrophic methanogens. These methanogens were able to withstand the relatively long aeration periods. Lowering the anaerobic solid retention time (SRT) from 17 to 8 days enabled selective removal of the methanogens and prevented unwanted methane formation. In absence of methanogens, methanol was converted aerobically, while granule formation remained stable. At high SRT-values (51 days) γ-Proteobacteria were responsible for acetate removal through anaerobic uptake and subsequent aerobic growth on storage polymers formed (so called metabolism of glycogen accumulating organisms). When lowering the SRT (24 days), Defluviicoccus-related organisms (cluster II) belonging to the α-Proteobacteria outcompeted acetate consuming γ-Proteobacteria at 35 ºC. DNA from the Defluviicoccus-related organisms in cluster II was not extracted by the standard DNA extraction method but with liquid nitrogen, which showed to be more effective. Remarkably, the two glycogen accumulating organisms (GAO) types of organisms grew separately in two clearly different types of granules. This work further highlights the potential of aerobic granular sludge systems to effectively influence the microbial communities through sludge age control in order to optimize the wastewater treatment processes. Recently, aerobic granular sludge technology has been scaled-up and implemented for industrial and municipal wastewater treatment under the trade name Nereda®. With full-scale references for industrial treatment application since 2006 and domestic sewage since 2009 only limited operating data have been presented in scientific literature so far. In this study performance, granulation and design considerations of an aerobic granular sludge plant on domestic wastewater at the WWTP Garmerwolde, the Netherlands were analysed (Chapter 6). After a start-up period of approximately 5 months, a robust and stable granule bed (> 8 g L-1) was formed and could be maintained thereafter, with a sludge volume index after 5 minutes settling of 45 mL g-1. The granular sludge consisted for more than 80 % of granules larger than 0.2 mm and more than 60 % larger than 1 mm. Effluent requirements (7 mg N L-1 and 1 mg P L-1) were easily met during summer and winter. Maximum volumetric conversion rates for nitrogen and phosphorus were respectively 0.17 and 0.24 kg (m3 d)-1. The energy usage was 13.9 kWh (PE150∙year)-1 that is 58 – 63 % lower than the average conventional activated sludge treatment plant in the Netherlands. Finally, this study demonstrated that aerobic granular sludge technology can effectively be implemented for the treatment of domestic wastewater. @en