Anionic extracellular polymeric substances extracted from seawater-adapted aerobic granular sludge.

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.

Microbiome structure and function in parallel full-scale aerobic granular sludge and activated sludge processes.

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. KEY POINTS: • Microbial community succession in parallel full-scale aerobic granular sludge (AGS) and conventional activated sludge (CAS) processes. • Higher periodicity in microbial community structure in CAS compared to in AGS. • Similar functional groups between AGS and CAS but different composition and dynamics at genus level.

Sialylation and Sulfation of Anionic Glycoconjugates Are Common in the Extracellular Polymeric Substances of Both Aerobic and Anaerobic Granular Sludges.

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.

Anaerobic hydrolysis of complex substrates in full-scale aerobic granular sludge: enzymatic activity determined in different sludge fractions.

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 ß-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.

Trehalose as an osmolyte in Candidatus Accumulibacter phosphatis.

Candidatus Accumulibacter phosphatis is an important microorganism for enhanced biological phosphorus removal (EBPR). In a previous study, we found a remarkable flexibility regarding salinity, since this same microorganism could thrive in both freshwater- and seawater-based environments, but the mechanism for the tolerance to saline conditions remained unknown. Here, we identified and described the role of trehalose as an osmolyte in Ca. Accumulibacter phosphatis. A freshwater-adapted culture was exposed to a single batch cycle of hyperosmotic and hypo-osmotic shock, which led to the release of trehalose up to 5.34 mg trehalose/g volatile suspended solids (VSS). Long-term adaptation to 30% seawater-based medium in a sequencing batch reactor (SBR) gave a stable operation with complete anaerobic uptake of acetate and propionate along with phosphate release of 0.73 Pmol/Cmol, and complete aerobic uptake of phosphate. Microbial analysis showed Ca. Accumulibacter phosphatis clade I as the dominant organism in both the freshwater- and seawater-adapted cultures (> 90% presence). Exposure of the seawater-adapted culture to a single batch cycle of hyperosmotic incubation and hypo-osmotic shock led to an increase in trehalose release upon hypo-osmotic shock when higher salinity is used for the hyperosmotic incubation. Maximum trehalose release upon hypo-osmotic shock was achieved after hyperosmotic incubation with 3× salinity increase relative to the salinity in the SBR adaptation reactor, resulting in the release of 11.9 mg trehalose/g VSS. Genome analysis shows the possibility of Ca. Accumulibacter phosphatis to convert glycogen into trehalose by the presence of treX, treY, and treZ genes. Addition of trehalose to the reactor led to its consumption, both during anaerobic and aerobic phases. These results indicate the flexibility of the metabolism of Ca. Accumulibacter phosphatis towards variations in salinity. KEY POINTS: • Trehalose is identified as an osmolyte in Candidatus Accumulibacter phosphatis. • Ca. Accumulibacter phosphatis can convert glycogen into trehalose. • Ca. Accumulibacter phosphatis clade I is present and active in both seawater and freshwater.

Importance of Species Sorting and Immigration on the Bacterial Assembly of Different-Sized Aggregates in a Full-Scale Aerobic Granular Sludge Plant.

In aerobic granular sludge (AGS) systems, different-sized microbial aggregates having different solids retention time (SRT) coexist in the same reactor compartment and are subjected to the same influent wastewater. Thus, the AGS system provides a unique ecosystem to study the importance of local (species sorting) and regional (immigration) processes in bacterial community assembly. The microbial communities of different-sized aggregates (flocs <0.2 mm, small granules (0.2-1.0 mm) and large granules >1.0 mm), influent wastewater, excess sludge and effluent of a full-scale AGS plant were characterized over a steady-state operation period of 6 months. Amplicon sequencing was integrated with mass balance to determine the SRT and net growth rate of operational taxonomic units (OTUs). We found strong evidence of species sorting as opposed to immigration, which was significantly higher at short SRT (i.e., flocs and small granules) than that at long SRT (large granules). Rare OTUs in wastewater belonging to putative functional groups responsible for nitrogen and phosphorus removal were progressively enriched with an increase in microbial aggregates size. In contrast, fecal- and sewage infrastructure-derived microbes progressively decreased in relative abundance with increase in microbial aggregate size. These findings highlight the importance of AGS as a unique model ecosystem to study fundamental microbial ecology concepts.

The water-soluble fraction of extracellular polymeric substances from a resource recovery demonstration plant: characterization and potential application as an adhesive.

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.

Demystifying polyphosphate-accumulating organisms relevant to wastewater treatment: A review of their phylogeny, metabolism, and detection.

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.

Effects of salinity on glycerol conversion and biological phosphorus removal by aerobic granular sludge.

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.

Alterations of Glycan Composition in Aerobic Granular Sludge during the Adaptation to Seawater Conditions.

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.

Utilizing anaerobic substrate distribution for growth of aerobic granular sludge in continuous-flow reactors.

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.

Density measurements of aerobic granular sludge.

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.

Enhancing phosphorus removal of photogranules by incorporating polyphosphate accumulating organisms.

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.

Aerobic granular sludge phosphate removal using glucose.

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, poly­hydroxy-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.

Metaproteomics, metagenomics and 16S rRNA sequencing provide different perspectives on the aerobic granular sludge microbiome.

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.

On the mechanisms for aerobic granulation - model based evaluation.

In this study a mathematical framework was developed to describe aerobic granulation based on 6 main mechanisms: microbial selection, selective wasting, maximizing transport of substrate into the biofilm, selective feeding, substrate type and breakage. A numerical model was developed using four main components; a 1D convection/dispersion model to describe the flow dynamics in a reactor, a reaction/diffusion model describing the essential conversions for granule growth, a setting model to track granules during settling and feeding, and a population model containing up to 100,000 clusters of granules to model the stochastic behaviour of the granulation process. With this approach the model can explain the dynamics of the granulation process observed in practice. This includes the presence of a lag phase and a granulation phase. Selective feeding was identified as an important mechanism that was not yet reported in literature. When aerobic granules are grown from activated sludge flocs, a lag phase occurs, in which not many granules are formed, followed by a granulation phase in which granules rapidly appear. The ratio of granule forming to non-granule forming substrate together with the feast/famine ratio determine if the transition from the lag phase to the granulation phase is successful. The efficiency of selective wasting and selective feeding both determine the rate of this transition. Brake-up of large granules into smaller well settling particles was shown to be an important source for new granules. The granulation process was found to be the combined result from all 6 mechanisms and if conditions for either one are not optimal, other mechanisms can, to some extent, compensate. This model provides a theoretical framework to analyse the different relevant mechanisms for aerobic granular sludge formation and can form the basis for a comprehensive model that includes detailed nutrient removal aspects.

Hydrolysis capacity of different sized granules in a full-scale aerobic granular sludge (AGS) reactor.

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.

Metagenomic profiling and transfer dynamics of antibiotic resistance determinants in a full-scale granular sludge wastewater treatment plant.

In the One Health context, wastewater treatment plants (WWTPs) are central to safeguarding water resources. Nonetheless, many questions remain about their effectiveness in preventing antimicrobial resistance (AMR) dissemination. Most surveillance studies monitor the levels and removal of selected antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in intracellular DNA (iDNA) extracted from WWTP influents and effluents. The role of extracellular free DNA (exDNA) in wastewater is mostly overlooked. This study analyzed the transfer of ARGs and MGEs in a full-scale Nereda® reactor removing nutrients with aerobic granular sludge. We tracked the composition and fate of the iDNA and exDNA pools of influent, sludge, and effluent samples. Metagenomics was used to profile the microbiome, resistome, and mobilome signatures of iDNA and exDNA extracts. Selected ARGs and MGEs were analyzed by qPCR. From 2,840 ARGs identified, the genes arr-3 (2%), tetC (1.6%), sul1 (1.5%), oqxB (1.2%), and aph(3")-Ib (1.2%) were the most abundant among all sampling points and bioaggregates. Pseudomonas, Acinetobacter, Aeromonas, Acidovorax, Rhodoferax, and Streptomyces populations were the main potential hosts of ARGs in the sludge. In the effluent, 478 resistance determinants were detected, of which 89% were from exDNA potentially released by cell lysis during aeration in the reactor. MGEs and multiple ARGs were co-localized on the same extracellular genetic contigs. Total intracellular ARGs decreased 3-42% due to wastewater treatment. However, the ermB and sul1 genes increased by 2 and 1 log gene copies mL-1, respectively, in exDNA from influent to effluent. The exDNA fractions need to be considered in AMR surveillance, risk assessment, and mitigation strategies.

Glycerol conversion by aerobic granular sludge.

Glycerol is abundantly present in wastewater from industries such as biodiesel production facilities. Glycerol is also a potential carbon source for microbes that are involved in wastewater nutrient removal processes. The conversion of glycerol in biological phosphorus removal of aerobic granular sludge processes has not been explored to date. The current study describes glycerol utilization by aerobic granular sludge and enhanced biological phosphorus removal (EBPR). Robust granules with good phosphorus removal capabilities were formed in an aerobic granular sludge sequencing batch reactor fed with glycerol. The interaction between the fermentative conversion of glycerol and product uptake by polyphosphate accumulating organisms (PAO) was studied using stoichiometric and microbial community analysis. Metagenomic, metaproteomic and microscopic analysis identified a community dominated by Actinobacteria (Tessaracoccus and Micropruina) and a typical PAO known as Ca. Accumulibacter. Glycerol uptake facilitator (glpF) and glycerol kinase (glpK), two proteins involved in the transport of glycerol into the cellular metabolism, were only observed in the genome of the Actinobacteria. The anaerobic conversion appeared to be a combination of a substrate fermentation and product uptake-type reaction. Initially, glycerol fermentation led mainly to the production of 1,3-propanediol (1,3-PDO) which was not taken up under anaerobic conditions. Despite the aerobic conversion of 1,3-PDO stable granulation was observed. Over time, 1,3-PDO production decreased and complete anaerobic COD uptake was observed. The results demonstrate that glycerol-containing wastewater can effectively be treated by the aerobic granular sludge process and that fermentative and polyphosphate accumulating organisms can form a food chain in glycerol-based EBPR processes.

Nitrous oxide emission from full-scale municipal aerobic granular sludge.

The nitrous oxides emission was measured over 7 months in the full-scale aerobic granular sludge plant in Dinxperlo, the Netherlands. Nitrous oxide concentrations were measured in the bulk liquid and the off-gas of the Nereda® reactor. Combined with the batch wise operation of the reactor, this gave a high information density and a better insight into N2O emission in general. The average emission factor was 0.33% based on the total nitrogen concentration in the influent. The yearly average emission factor was estimated to be between 0.25% and 0.30%. The average emission factor is comparable to continuous activated sludge plants, using flocculent sludge, and it is low compared to other sequencing batch systems. The variability in the emission factor increased when the reactor temperature was below 14 °C, showing higher emission factors during the winter period. A change in the process control in the winter period reduced the variability, reducing the emission factors to a level comparable to the summer period. Different process control might be necessary at high and low temperatures to obtain a consistently low nitrous oxide emission. Rainy weather conditions lowered the emission factor, also in the dry weather flow batches following the rainy weather batches. This was attributed to the first flush from the sewer at the start of rainy weather conditions, resulting in a temporarily increased sludge loading.