Aerobic granular sludge (AGS) technology holds great promise of becoming the standard for biological wastewater treatment due to its lower energy consumption, small footprint, and high removal efficiency of nutrients compared to the conventional activated sludge processes. Different-sized aggregates have been shown to harbor a different microbial community composition. The central question is do full-scale AGS wastewater treatment plants (WWTPs) select for core microbial communities across different aggregate sizes and how these selected organisms differ between the different-sized aggregates. This study analyzed samples from nine geographically distributed full-scale AGS WWTPs that consistently perform well in terms of chemical oxygen demand (COD) and nutrient (N and P) removal. The main results showed that site-specific conditions highly influence microbial composition in smaller aggregates (< 1 mm), while larger granules form stable communities independent of WWTP location. Notably, all aggregates contained a small subset of 128–139 core OTUs that were both prevalent and abundant across all sizes. These core OTUs include key functional groups such as fermenters, aerobic heterotrophs, polyphosphate-accumulating organisms (PAOs), glycogen-accumulating organisms (GAOs), and nitrifiers, which play a crucial role in COD and nutrient removal. Additionally, an enrichment pattern was observed, with aerobic heterotrophs dominating in flocs, PAOs in small granules, and GAOs and nitrifiers in large granules. This study offers valuable insights into the core microbiome of different-sized aggregates in full-scale AGS WWTPs and highlights their potential role in overall system performance.

Understanding the relative influence of immigration and species sorting in wastewater treatment systems is essential, as bacteria in influent wastewater can significantly impact treatment system functionality. This study investigated the contribution of immigration to the community assembly of different-sized microbial aggregates in a full-scale aerobic granular sludge (AGS) system using genome-resolved metatranscriptomics. Our novel analysis revealed that negative-net-growth-rate populations, which persist due to immigration, can exhibit substantial activity and potentially contribute to the AGS system’s functionality. The results also highlighted that sulfate-reducing and fermenting bacteria, along with some nitrifiers and glycogen-accumulating organisms (GAOs), were more active in the influent wastewater, serving as a continuous source of both beneficial and competing immigrants to the AGS system. Granular sludge (size >0.2 mm) demonstrated a robust capacity to resist immigration effects from competing immigrants, whereas flocculent sludge (size <0.2 mm) was more susceptible. Importantly, flocculent sludge harbored functional microbial groups such as active nitrifiers and fermentative polyphosphate-accumulating organisms (PAOs) belonging to Ca. Phosphoribacter, while granular sludge enriched for active conventional PAOs such as Ca. Accumulibacter. These findings provide valuable insights for engineers to design and operate AGS systems by optimizing microbial aggregate sizes and emphasizing the importance of influent microbial characterization in the design of wastewater treatment plants to enhance the functionality and activity of AGS systems.

Microbial aggregates of different sizes in aerobic granular sludge (AGS) systems have been shown to exhibit distinct microbial community compositions. However, studies comparing the microbial activities of different-sized aggregates in AGS systems remain limited. In this study, genome-resolved metatranscriptomics was used to investigate microbial activity patterns within differently sized aggregates in a full-scale AGS plant. Our analysis revealed a weak correlation between the relative abundance of metagenome-assembled genomes (MAGs) and their transcriptomic activity, indicating that microbial abundance does not directly correspond to metabolic activity within the system. Flocculent sludge (FL; <0.2 mm) predominantly featured active nitrifiers and fermentative polyphosphate-accumulating organisms (PAOs) from Candidatus Phosphoribacter, while small granules (SG; 0.2–1.0 mm) and large granules (LG; >1.0 mm) hosted more metabolically active PAOs affiliated with Ca. Accumulibacter. Differential gene expression analysis further supported these findings, demonstrating significantly higher expression levels of key phosphorus uptake genes associated with Ca. Accumulibacter in granular sludge (SG and LG) compared to flocculent sludge. Conversely, Ca. Phosphoribacter showed higher expression of these genes in the FL fraction. This study highlights distinct functional roles and metabolic activities of crucial microbial communities depending on aggregate size within AGS systems, offering new insights into optimizing wastewater treatment processes.

The development of continuous flow reactors (CFRs) employing aerobic granular sludge (AGS) for the retrofit of existing wastewater treatment plants (WWTPs) using a continuous-flow activated sludge (CFAS) system has garnered increasing interest. This follows the worldwide adoption of AGS technology in sequencing batch reactors (SBRs). The better settleability of AGS compared to AS allows for process intensification of existing wastewater treatment plants without the difficult conversion of often relatively shallow CFRs to deeper AGS-SBRs. To retrofit existing CFAS systems with AGS, achieving both increased hydraulic capacity and enhanced biological nutrient removal necessitates the formation of granular sludge based on the same selective pressures applied in AGS-SBRs. Previous efforts have focussed mainly on the selective wasting of flocculent sludge and retaining granular sludge to drive aerobic granulation. In this study a pilot-scale CFR was developed to best mimic the implementation of the granulation mechanisms of full-scale AGS-SBRs. The pilot-scale reactor was fed with pre-settled municipal wastewater. We established metrics to assess the degree to which the proposed mechanisms were implemented in the pilot-scale CFR and compared them to data from full-scale AGS-SBRs, specifically with respect to the anaerobic distribution of granule forming substrates (GFS). The selective pressures for granular sludge formation were implemented through inclusion of anaerobic upflow selectors with a water depth of 2.5 meters, which yielded a sludge with properties similar to AGS from full-scale SBRs. In comparison to the CFAS system at Harnaschpolder WWTP treating the same pre-settled wastewater, a more than twofold increase in volumetric removal capacity for both phosphorus and nitrogen was achieved. The use of a completely mixed anaerobic selector, as opposed to an anaerobic upflow selector, caused a shift in EBPR activity from the largest towards the smallest size class, while nitrification was majorly unaffected. Anaerobic selective feeding via bottom-feeding is, therefore, favorable for the long-term stability of AGS, especially for less acidified wastewater. The research underlines the potential of AGS for enhancing the hydraulic and biological treatment capacity of existing CFAS systems.

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.

Industrial wastewater often has high levels of salt, either due to seawater or e.g. sodium chloride (NaCl) usage in the processing. Previous work indicated that aerobic granular sludge (AGS) is differently affected by seawater or saline water at similar osmotic strength. Here we investigate in more detail the impact of NaCl concentrations and seawater on the granulation and conversion processes for AGS wastewater treatment. Glycerol was used as the carbon source since it is regularly present in industrial wastewaters, and to allow the evaluation of microbial interactions that better reflect real conditions. Long-term experiments were performed to evaluate and compare the effect of salinity on granulation, anaerobic conversions, phosphate removal, and the microbial community. Smooth and stable granules as well as enhanced biological phosphorus removal (EBPR) were achieved up to 20 g/L NaCl or when using seawater. However, at NaCl levels comparable to seawater strength (30 g/L) incomplete anaerobic glycerol uptake and aerobic phosphate uptake were observed, the effluent turbidity increased, and filamentous granules began to appear. The latter is likely due to the direct aerobic growth on the leftover substrate after the anaerobic feeding period. In all reactor conditions, except the reactor with 30 g/L NaCl, Ca. Accumulibacter was the dominant microorganism. In the reactor with 30 g/L NaCl, the relative abundance of Ca. Accumulibacter decreased to ≤1 % and an increase in the genus Zoogloea was observed. Throughout all reactor conditions, Tessaracoccus and Micropruina, both actinobacteria, were present which were likely responsible for the anaerobic conversion of glycerol into volatile fatty acids. None of the glycerol metabolizing proteins were detected in Ca. Accumulibacter which supports previous findings that glycerol can not be directly utilized by Ca. Accumulibacter. The proteome profile of the dominant taxa was analysed and the results are further discussed. The exposure of salt-adapted biomass to hypo-osmotic conditions led to significant trehalose and PO₄³⁻-P release which can be related to the osmoregulation of the cells. Overall, this study provides insights into the effect of salt on the operation and stability of the EBPR and AGS processes. The findings suggest that maintaining a balanced cation ratio is likely to be more important for the operational stability of EBPR and AGS systems than absolute salt concentrations.

Currently, there is a growing interest in transforming wastewater treatment plants (WWTPs) into resource recovery plants. Microorganisms in aerobic granular sludge produce extracellular polymeric substances (EPS), which are considered sustainable resources to be extracted and can be used in diverse applications. Exploring applications in other high-value materials, such as adhesives, will not only enhance the valorization potential of the EPS but also promote resource recovery. This study aimed to characterize a water-soluble fraction extracted from the EPS collected at the demonstration plant in the Netherlands based on its chemical composition (amino acids, sugar, and fatty acids) and propose a proof-of-concept for its use as an adhesive. This fraction comprises a mixture of biomolecules, such as proteins (26.6 ± 0.3%), sugars (21.8 ± 0.2%), and fatty acids (0.9%). The water-soluble fraction exhibited shear strength reaching 36–51 kPa across a pH range of 2–10 without additional chemical treatment, suggesting a potential application as an adhesive. The findings from this study provide insights into the concept of resource recovery and the valorization of excess sludge at WWTPs.

Aerobic granular sludge (AGS) and conventional activated sludge (CAS) are two different biological wastewater treatment processes. AGS consists of self-immobilised microorganisms that are transformed into spherical biofilms, whereas CAS has floccular sludge of lower density. In this study, we investigated the treatment performance and microbiome dynamics of two full-scale AGS reactors and a parallel CAS system at a municipal WWTP in Sweden. Both systems produced low effluent concentrations, with some fluctuations in phosphate and nitrate mainly due to variations in organic substrate availability. The microbial diversity was slightly higher in the AGS, with different dynamics in the microbiome over time. Seasonal periodicity was observed in both sludge types, with a larger shift in the CAS microbiome compared to the AGS. Groups important for reactor function, such as ammonia-oxidising bacteria (AOB), nitrite-oxidising bacteria (NOB), polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs), followed similar trends in both systems, with higher relative abundances of PAOs and GAOs in the AGS. However, microbial composition and dynamics differed between the two systems at the genus level. For instance, among PAOs, Tetrasphaera was more prevalent in the AGS, while Dechloromonas was more common in the CAS. Among NOB, Ca. Nitrotoga had a higher relative abundance in the AGS, while Nitrospira was the main nitrifier in the CAS. Furthermore, network analysis revealed the clustering of the various genera within the guilds to modules with different temporal patterns, suggesting functional redundancy in both AGS and CAS.

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.

Abstract: Anionic polymers, such as heparin, have been widely applied in the chemical and medical fields, particularly for binding proteins (e.g., fibroblast growth factor 2 (FGF-2) and histones). However, the current animal-based production of heparin brings great risks, including resource shortages and product contamination. Recently, anionic compounds, nonulosonic acids (NulOs), and sulfated glycoconjugates were discovered in the extracellular polymeric substances (EPS) of aerobic granular sludge (AGS). Given the prevalence of anionic polymers, in marine biofilms, it was hypothesized that the EPS from AGS grown under seawater condition could serve as a raw material for producing the alternatives to heparin. This study aimed to isolate and enrich the anionic fractions of EPS and evaluate their potential application in the chemical and medical fields. The AGS was grown in a lab-scale reactor fed with acetate, under the seawater condition (35 g/L sea salt). The EPS was extracted with an alkaline solution at 80 °C and fractionated by size exclusion chromatography. Its protein binding capacity was evaluated by native gel electrophoresis. It was found that the two highest molecular weight fractions (438– > 14,320 kDa) were enriched with NulO and sulfate-containing glycoconjugates. The enriched fractions can strongly bind the two histones involved in sepsis and a model protein used for purification by heparin-column. These findings demonstrated possibilities for the application of the extracted EPS and open up a novel strategy for resource recovery. Key points: • High MW EPS from seawater-adapted AGS are dominant with sulfated groups and NulOs • Fifty-eight percent of the EPS is high MW of 68–14,320 kDa • EPS and its fractions can bind histones and fibroblast growth factor 2 Graphical Abstract: [Figure not available: see fulltext.]

There is a growing interest to implement aerobic granular sludge (AGS) in existing conventional activated sludge (CAS) systems with a continuous flow-through configuration. The mode of anaerobic contact of raw sewage with the sludge is an important aspect in the adaptation of CAS systems to accommodate AGS. It remains unclear how the distribution of substrate over the sludge by a conventional anaerobic selector compares to the distribution via bottom-feeding applied in sequencing batch reactors (SBRs). This study investigated the effect of the anaerobic contact mode on the substrate (and storage) distribution by operating two lab-scale SBRs; one with the traditional bottom-feeding through a settled sludge bed similar to full-scale AGS systems, and one where the synthetic wastewater was fed as a pulse at the start of the anaerobic phase while the reactor was mixed through sparging of nitrogen gas (mimicking a plug-flow anaerobic selector in continuous flow-through systems). The distribution of the substrate over the sludge particle population was quantified via PHA analysis, combined with the obtained granule size distribution. Bottom-feeding was found to primarily direct substrate towards the large granular size classes (i.e. large volume and close to the bottom), while completely mixed pulse-feeding gives a more equal distribution of substrate over all granule sizes (i.e. surface area dependant). The anaerobic contact mode directly controls the substrate distribution over the different granule sizes, irrespective of the solids retention time of a granule as an entity. Preferential feeding of the larger granules will enhance and stabilise the granulation compared to pulse-feeding, certainly under less advantageous conditions imposed by real sewage.

Photogranules are a novel wastewater treatment technology that can utilize the sun's energy to treat water with lower energy input and have great potential for nutrient recovery applications. They have been proven to efficiently remove nitrogen and carbon but show lower conversion rates for phosphorus compared to established treatment systems, such as aerobic granular sludge. In this study, we successfully introduced polyphosphate accumulating organisms (PAOs) to an established photogranular culture. We operated photobioreactors in sequencing batch mode with six cycles per day and alternating anaerobic (dark) and aerobic (light) phases. We were able to increase phosphorus removal/recovery by 6 times from 5.4 to 30 mg/L/d while maintaining similar nitrogen and carbon removal compared to photogranules without PAOs. To maintain PAOs activity, alternating anaerobic feast and aerobic famine conditions were required. In future applications, where aerobic conditions are dependent on in-situ oxygenation via photosynthesis, the process will rely on sunlight availability. Therefore, we investigated the feasibility of the process under diurnal cycles with a 12-h anaerobic phase during nighttime and six short cycles during the 12 h daytime. The 12-h anaerobic phase had no adverse effect on the PAOs and phototrophs. Due to the extension of one anaerobic phase to 12 h the six aerobic phases were shortened by 47% and consequently decreased the light hours per day. This resulted in a decrease of phototrophs, which reduced nitrogen removal and biomass productivity up to 30%. Finally, we discuss and suggest strategies to apply PAO-enriched photogranules at large-scale.

Bacteria can synthesize a diverse array of glycans, being found attached to proteins and lipids or as loosely associated polysaccharides to the cells. The major challenge in glycan analysis in environmental samples lies in developing high-throughput and comprehensive characterization methodologies to elucidate the structure and monitor the change of the glycan profile, especially in protein glycosylation. To this end, in the current research, the dynamic change of the glycan profile of a few extracellular polymeric substance (EPS) samples was investigated by high-throughput lectin microarray and mass spectrometry, as well as sialylation and sulfation analysis. Those EPS were extracted from aerobic granular sludge collected at different stages during its adaptation to the seawater condition. It was found that there were glycoproteins in all of the EPS samples. In response to the exposure to seawater, the amount of glycoproteins and their glycan diversity displayed an increase during adaptation, followed by a decrease once the granules reached a stable state of adaptation. Information generated sheds light on the approaches to identify and monitor the diversity and dynamic alteration of the glycan profile of the EPS in response to environmental stimuli.

The tremendous progress in sequencing technologies has made DNA sequencing routine for microbiome studies. Additionally, advances in mass spectrometric techniques have extended conventional proteomics into the field of microbial ecology. However, systematic studies that provide a better understanding of the complementary nature of these 'omics' approaches, particularly for complex environments such as wastewater treatment sludge, are urgently needed. Here, we describe a comparative metaomics study on aerobic granular sludge from three different wastewater treatment plants. For this, we employed metaproteomics, whole metagenome, and 16S rRNA amplicon sequencing to study the same granule material with uniform size. We furthermore compare the taxonomic profiles using the Genome Taxonomy Database (GTDB) to enhance the comparability between the different approaches. Though the major taxonomies were consistently identified in the different aerobic granular sludge samples, the taxonomic composition obtained by the different omics techniques varied significantly at the lower taxonomic levels, which impacts the interpretation of the nutrient removal processes. Nevertheless, as demonstrated by metaproteomics, the genera that were consistently identified in all techniques cover the majority of the protein biomass. The established metaomics data and the contig classification pipeline are publicly available, which provides a valuable resource for further studies on metabolic processes in aerobic granular sludge.

Anaerobic and aerobic granular sludge processes are widely applied in wastewater treatment. In these systems, microorganisms grow in dense aggregates due to the production of extracellular polymeric substances (EPS). This study investigates the sialylation and sulfation of anionic glyconconjugates in anaerobic and aerobic granular sludges collected from full-scale wastewater treatment processes. Size exclusion chromatography revealed a wide molecular weight distribution (3.5 to >5500 kDa) of the alkaline-extracted EPS. The high-molecular weight fraction (>5500 kDa), comprising 16.9-27.4% of EPS, was dominant with glycoconjugates. Mass spectrometry analysis and quantification assays identified nonulosonic acids (NulOs, e.g., bacterial sialic acids) and sulfated groups contributing to the negative charge in all EPS fractions. NulOs were predominantly present in the high-molecular weight fraction (47.2-84.3% of all detected NulOs), while sulfated glycoconjugates were distributed across the molecular weight fractions. Microorganisms, closely related to genera found in the granular sludge communities, contained genes responsible for NulO and sulfate group synthesis or transfer. The similar distribution patterns of sialylation and sulfation of the anionic glycoconjugates in the EPS samples indicate that these two glycoconjugate modifications commonly occur in the EPS of aerobic and anaerobic granular sludges.

Enhanced biological phosphate removal and aerobic sludge granulation are commonly studied with fatty acids as substrate. Fermentative substrates such as glucose have received limited attention. In this work, glucose conversion by aerobic granular sludge and its impact on phosphate removal was studied. Long-term stable phosphate removal and successful granulation were achieved. Glucose was rapidly taken up (273 mg/gVSS/h) at the start of the anaerobic phase, while phosphate was released during the full anaerobic phase. Some lactate was produced during glucose consumption, which was anaerobically consumed once glucose was depleted. The phosphate release appeared to be directly proportional to the uptake of lactate. The ratio of phosphorus released to glucose carbon taken up over the full anaerobic phase was 0.25 Pmol/Cmol. Along with glucose and lactate uptake in the anaerobic phase, poly‑hydroxy-alkanoates and glycogen storage were observed. There was a linear correlation between glucose consumption and lactate formation. While lactate accounted for approximately 89 % of the observed products in the bulk liquid, minor quantities of formate (5 %), propionate (4 %), and acetate (3 %) were also detected (mass fraction). Formate was not consumed anaerobically. Quantitative fluorescence in-situ hybridization (qFISH) revealed that polyphosphate accumulating organisms (PAO) accounted for 61 ± 15 % of the total biovolume. Metagenome evaluation of the biomass indicated a high abundance of Micropruina and Ca. Accumulibacter in the system, which was in accordance with the microscopic observations and the protein mass fraction from metaproteome analysis. Anaerobic conversions were evaluated based on theoretical ATP balances to provide the substrate distribution amongst the dominant genera. This research shows that aerobic granular sludge technology can be applied to glucose-containing effluents and that glucose is a suitable substrate for achieving phosphate removal. The results also show that for fermentable substrates a microbial community consisting of fermentative organisms and PAO develop.

This study describes a novel integration of aerobic granular sludge (AGS) with a gravity-driven membrane (GDM) system at a pilot scale with a treatment capacity of approximately 150 L per day to treat raw domestic wastewater. The treatment performance and energy consumption of the AGS-GDM system were compared to the neighboring full-scale aerobic membrane bioreactor (AeMBR), treating the same wastewater at about 4000(±500) m³ per day. The AGS-GDM system demonstrated superior nutrient (nitrogen and phosphorus) removal as compared to the AeMBR. The GDM unit was continuously supplied with AGS-treated effluent. The GDM unit started with high [ >20 L per m² per h (LMH) ] flux, which gradually declined. The flux remained quite stable after 15 days reaching 3 LMH after 35 days without any physical or chemical cleaning. Our results suggest that AGS-GDM is a viable technology for decentralized wastewater treatment and reuse in water-scarce regions. The AGS-GDM could easily replace conventional AeMBR technology in the wastewater treatment and reclamation market.

Granular sludge processes are frequently used in domestic and industrial wastewater treatment. The granule buoyant density and biomass density are important parameters for the design and operation of granular sludge reactors. Different methods to measure the granule density include the pycnometer method, the Percoll density gradient method, the dextran blue method, and the settling velocity method. In this study, a comparison was made between these four methods to measure granule density for granules from a full-scale granular sludge plant treating domestic sewage. The effect of salinity on granule density was assessed as well. Three out of the four evaluated methods yielded comparable results, with granule buoyant densities between 1025.7 and 1028.1 kg/m³ and granule biomass densities between 71.1 and 71.5 g/L (based on volatile suspended solids (VSS)). The settling velocity method clearly underestimated the granule density, due to the complex relation between granule properties and settling velocity. The pycnometer method was the most precise method, but it was also quite susceptible to bias. The granule buoyant density increased proportionally with salinity, to 1049.2 kg/m³ at 36 g/L salinity. However, the granule biomass density, based on VSS, remained constant. This showed that the granule volume was not affected by salinity and that the buoyant density increase was the result of diffusion of salts into the granule pores. Overall, the granule density can be measured reliably with most methods, as long as the effect of salinity is considered. The results are discussed in light of operational aspects for full-scale granular sludge plants.

In aerobic granular sludge (AGS) reactors, granules of different sizes coexist in a single reactor. Their differences in settling behaviour cause stratification in the settled granule bed. In combination with substrate concentration gradients over the reactor height during the anaerobic plug-flow feeding regime, this can result in functional differences between granule sizes. In this study, we compared the hydrolytic activity in granules of 4 size ranges (between 0.5 and 4.8 mm diameter) collected from a full-scale AGS installation. Protease and amylase activities were quantified through fluorescent activity assays. To visualise where the hydrolytic active zones were located within the granules, the hydrolysis sites were visualized microscopically after incubating intact and sliced granules with fluorescent casein and starch. The microbial community was studied using fluorescent in situ hybridization (FISH) and sequencing. The results of these assays indicated that hydrolytic capacity was present throughout the granules, but the hydrolysis of bulk substrates was restricted to the outer 100 µm, approximately. Many of the microorganisms studied by FISH, such as polyphosphate and glycogen accumulating organisms (PAO and GAO), were abundant in the vicinity of the hydrolytically active sites. The biomass-specific hydrolysis rate depended mainly on the available granule surface area, suggesting that different sized granules are not differentiated in terms of hydrolytic capacity. Thus, the substrate concentration gradients that are present during the anaerobic feeding in AGS reactors do not seem to affect hydrolytic activity at the granule surfaces. In this paper, we discuss the possible reasons for this and reflect about the implications for AGS technology.