Identifying structural proteins within the extracellular polymeric substances (EPS) will provide a better understanding of the stability of aerobic granular sludge (AGS) and biofilms in general. In this work, an abundant surface protein was identified and localized in the extracellular matrix of seawater-adapted AGS. Granules with good phosphate removal were cultivated in a sequencing batch bubble column reactor with acetate as a carbon source dissolved in seawater. “Candidatus Accumulibacter” was observed as the most dominant community member through fluorescent in-situ hybridization. A surface protein of 74.5 kDa was identified in the EPS extract of the seawater-adapted AGS by SDS-PAGE and mass spectrometry. The surface protein was produced by an Accumulibacter species and showed homology to S-layer proteins. A type 1 secretion system was found adjacent to the gene encoding for the surface protein, suggesting a possible export system. Antibodies generated from a unique peptide of the surface protein confirmed the extracellular location of the surface protein. Microscopy observations with antibody staining showed the surface protein forms dense structures within the Accumulibacter microcolonies and larger fiber structures around the microcolonies. These observations highlight the importance of the protein for the structural properties of the granule. To detect more structural proteins in the EPS, optimization of the EPS extraction and in situ imaging validation methods are essential.

Limited understanding of the thermal properties and flammability of extracellular polymeric substances (EPS) from municipal sludge has constrained their use in fire safety applications. This study investigates the thermal stability and flame retardancy of EPS derived from secondary sludge, waste sludge, and digested sludges. Comprehensive analyses of chemical composition, activation energy, char formation, and heat and gas release during pyrolysis and combustion reveal potential flame-retardant mechanisms. All EPS from different types of sludge can release both free-radical scavenging compounds and non-flammable gases during combustion to reduce fire propagation in the gas phase. In the condensed phase, EPS demonstrate their char-forming capability as a major flame-retardant mechanism to diminish heat release rate due to the presence of nitrogen, phosphorus, and sulfur elements. While nitrogen-based compounds in EPS contribute to flame-retardant mechanisms in both phases, phosphorus and sulfur mainly act in the condensed phase. Additionally, EPS from secondary sludge show the highest activation energy (372.3 kJ/mol) and ignite 24 s later than other EPS. Cone calorimeter tests confirm that EPS from secondary sludge have superior fire-resistant properties, with increased char contents (31.2 %) and enhanced thermal stability compared to other EPS. This study provides the first comprehensive assessment of EPS as eco-friendly flame retardants, advancing wastewater sludge valorisation and fire safety applications.

Extracellular Polymeric Substances (EPS) are ubiquitous in biological wastewater treatment (WWT) technologies like activated sludge systems, biofilm reactors, and granular sludge systems. EPS recovery from sludge potentially offers a high-value material for the industry. It can be utilized as a coating in slow-release fertilizers, as a bio-stimulant, as a binding agent in building materials, for the production of flame retarding materials, and more. P recovered within the extracted EPS is an intrinsic part of the recovered material that potentially influences its properties and industrial applications. P is present in EPS in different speciation (e.g., P esters, poly-P, ortho-P, etc.). Such P species are already intensively used in the chemical industry to enhance thermal stability, viscoelasticity, emulsification, water-holding capacity, and many other properties of some natural and petroleum-derived polymers. The translation of this knowledge to EPS is missing which prevents the full utilization of phosphorus in EPS. This knowledge could allow us to engineer EPS via phosphorus for specific target properties and applications. In this review, we discuss how P could affect EPS properties based on experiences from other industries and reflect on how these P species could be influenced during the EPS extraction process or in the WWTPs.

Various methods for recovering extracellular polymeric substances (EPS)-based biomaterials from wastewater sludge exist. However, the relationships between extraction methods and properties of biomaterials have not been fully explored. In this study, the thermal properties, including activation energy (AE) and thermal decomposition mechanism, of EPS-based biomaterials extracted by different methods have been determined by thermogravimetric analysis integrated with the deconvolution method. Simultaneously, the chemical properties of these biomaterials, such as the extraction yield, chemical composition, and functional groups, have been monitored to clarify the influences of extraction methods. Notably, proteins and humic-like substances have been found as the major components to determine thermal stability and AE. Moreover, the physicochemical method shows significant effects on enhancing extraction yield and AE, with the NaOH and heat methods proving to be outstanding by delivering the highest AE of 300 kJ/mol and a substantial char formation of 24%. The results have demonstrated the significant impact of extraction methods on the thermal stability of EPS-based biomaterials. Moreover, this finding provides insights into the linkages between the properties of EPS-based biomaterials and extraction methods to guide the selection of appropriate extraction methods tailored to specific applications, including flame-resistant materials.

An interesting and potential property of extracellular polymeric substances (EPS) is the hydrogel formation with calcium ions. Aiming at understanding the significant difference in the hydrogel formed between EPS from flocculent and granular sludge, a targeted investigation of the lipopolysaccharides (LPS), one of the important EPS components, was performed. LPS was isolated from the EPS of flocculent and granular sludge, and both the glycan and the lipid A parts of LPS were characterized and compared. The morphology of LPS-calcium (LPS-Ca) aggregates were visualized by the polymyxin B-based fluorescent probe. The LPS constituted about 25 % and 15 % of the EPS from flocculent and granular sludge, respectively. The flocculent sludge LPS showed a lower amount of glycans, shorter glycan chain length, lower molecular weight, and higher possibility of containing unsaturated lipids than the granular sludge EPS. The flocculent sludge LPS-Ca aggregates demonstrated invert structures with the water phase in between, contributing to the fluid-like property of the respect EPS-Ca. In contrast, with the remarkably different chemical structure, LPS-Ca aggregates from granular sludge displayed bilaminar multilayered morphology, contributing to the solid, self-standing hydrogel of EPS-Ca.

Biological wastewater treatment relies on microorganisms that grow as flocs, biofilms, or granules for efficient separation of biomass from cleaned water. This biofilm structure emerges from the interactions between microbes that produce, and are embedded in, extracellular polymeric substances (EPS). The true composition and structure of the EPS responsible for dense biofilm formation are still obscure. We conducted a bottom-up approach utilizing advanced glycomic techniques to explore the glycan diversity in the EPS from a highly enriched “Candidatus Accumulibacter” granular sludge. Rare novel sugar monomers such as N-Acetylquinovosamine (QuiNAc) and 2-O-Methylrhamnose (2-OMe-Rha) were identified to be present in the EPS of both enrichments. Further, a high diversity in the glycoprotein structures of said EPS was identified by means of lectin based microarrays. We explored the genetic potential of “Ca. Accumulibacter” high quality metagenome assembled genomes (MAGs) to showcase the shortcoming of top-down bioinformatics based approaches at predicting EPS composition and structure, especially when dealing with glycans and glycoconjugates. This work suggests that more bottom-up research is necessary to understand the composition and complex structure of EPS in biofilms since genome based inference cannot directly predict glycan structures and glycoconjugate diversity.

Biofilms play important roles in water technologies such as membrane treatments and activated sludge. The extracellular polymeric substances (EPS) are key components of biofilms. However, the precise nature of these substances and how they influence biofilm formation and behavior remain critical knowledge gaps. EPS are produced by many different microorganisms and span multiple biopolymer classes, which each require distinct strategies for characterization. The biopolymers additionally associate with each other to form insoluble complexes. Here, we explore recent progress toward resolving the structures and functions of EPS, where a shift towards direct functional assessments and advanced characterization techniques is necessary. This will enable integration with better microbial community and omics analyses to understand EPS biosynthesis pathways and create further opportunities for EPS control and valorization.

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.

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.

Glycans are crucial for the structure and function of anaerobic granular sludge in wastewater treatment. Yet, there is limited knowledge regarding the microorganisms and biosynthesis pathways responsible for glycan production. In this study, we analysed samples from anaerobic granular sludges treating papermill and brewery wastewater, examining glycans composition and using metagenome-assembled genomes (MAGs) to explore potential biochemical pathways associated with their production. Uronic acids were the predominant constituents of the glycans in extracellular polymeric substances (EPS) produced by the anaerobic granular sludges, comprising up to 60 % of the total polysaccharide content. MAGs affiliated with Anaerolineacae, Methanobacteriaceae and Methanosaetaceae represented the majority of the microbial community (30–50 % of total reads per MAG). Based on the analysis of MAGs, it appears that Anaerolinea sp. and members of the Methanobacteria class are involved in the production of exopolysaccharides within the analysed granular sludges. These findings shed light on the functional roles of microorganisms in glycan production in industrial anaerobic wastewater treatment systems.

Over the past decade, a significant amount of research work on extracellular polymeric substances has been done on the “alginate-like exopolysaccharides” (ALE, also called “alginate-like exopolymers”). The term was used based on the FAO (Food and Agriculture Organization) biopolymer identification test. Although various chemical analyses have been conducted to characterize extracted ALE, it remained unclear whether ALE contains the two sugar monomers of alginates. Aiming to obtain a direct answer to the question: are there alginates in the ALE extracted from sludge, activated sludge was collected from two wastewater treatment plants in two different countries, where the ALE was previously extracted, characterized, and reported in the literature. The extracellular polymers were extracted from these sludges and fractionated according to the standard protocol. The sugar monomer composition of each fraction was analyzed, with special attention to the presence of mannuronic acid (M) and guluronic acid (G), which compose alginate biopolymers. None of these monomers were found in the extracted EPS, indicating there are no alginates resembling polymers extracted from the sludges. The possibility of the presence of other glycan components, such as lipopolysaccharides in EPS, was investigated and confirmed.

Climate change–driven sea level rise threatens freshwater ecosystems and elicits salinity stress in microbiomes. Methane emissions in these systems are largely mitigated by methane-oxidizing microorganisms. Here, we characterized the physiological and metabolic response of freshwater methanotrophic archaea to salt stress. In our microcosm experiments, inhibition of methanotrophic archaea started at 1%. However, during gradual increase of salt up to 3% in a reactor over 12 weeks, the culture continued to oxidize methane. Using gene expression profiles and metabolomics, we identified a pathway for salt-stress response that produces the osmolyte of anaerobic methanotrophic archaea: N(ε)-acetyl-β-L-lysine. An extensive phylogenomic analysis on N(ε)-acetyl-β-L-lysine-producing enzymes revealed that they are widespread across both bacteria and archaea, indicating a potential horizontal gene transfer and a link to BORG extrachromosomal elements. Physicochemical analysis of bioreactor biomass further indicated the presence of sialic acids and the consumption of intracellular polyhydroxyalkanoates in anaerobic methanotrophs during salt stress.

As a significant structure in activated sludge, extracellular polymeric substances (EPS) hold considerable value regarding resource recovery and applications. The present study aimed to elucidate the relationship between the microbial community and the composition and properties of EPS. A biological nutrient removal (BNR) reactor was set up in the laboratory and controlled under different solid retention times (SRT), altering microbial species within the system. Then EPS was extracted from activated and analyzed by chemical and spectroscopic methods. High-throughput sequencing and metagenomic approaches were employed to investigate bacterial community and metabolic pathways. The results showed that lower SRT with a higher abundance of the family-level Proteobacteria (27.7%-53.5%) favored EPS synthesis, while another dominant group Bacteroidetes (20.0%-32.6%) may not significantly affect EPS synthesis. Furthermore, the abundance of alginates-producing bacteria including Pseudomonas spp. and Azotobacter vinelandii was only 2.53%-6.76% and 1.98%-6.34%, respectively. The alginate synthesis pathway genes Alg8 and Alg44 were also present at very low levels (0.05‱-0.11‱, 0.01‱-0.02‱, respectively). Another important gene related to alginates operons, AlgK, was absent across all the SRT-operated reactors. These findings suggest an impossible and incomplete alginate synthesis pathway within sludge. In light of these results, it can be concluded that EPS does not necessarily contain alginate components.

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.

Sediment formation in drinking water distribution systems can lead to brown water at customer taps. Previous studies have shown that sediment formation is closely linked with (micro)biological processes in the distribution system, however the mechanism is not fully understood. Most available studies on discoloration or sediment formation mechanism are based on modeling, pilot-scale experiments, or low frequency data collected during pipe flushing. In this study, long-term sediment development in a large-scale drinking water distribution system was studied at one location over 11 years and at several locations along a known water trajectory during one year. Particulate material was collected at several locations using built-in and mobile filters that were connected to transport and distribution pipes in a semi-continuous manner. The volume of the collected material varied seasonally and the highest volumes were collected in the summer season. The material followed similar variations as temperature, invertebrates biomass and concentration of Aeromonas. The results showed that particulate matter of the sediment at downstream distribution locations was not released by the treatment works but instead forms along the distribution network, with increasing particle/floc size, biomass and Fe and Mn content. The large crustacean, Asellus, contributed to material production through feces excretion and formation of detritus by degradation of exoskeletons of dead animals. Detailed chemical characterization of the collected material showed the presence of proteins, calcium carbonate and iron precipitates. A similar sediment composition in a reference distribution system where customer complaints about brown water are experienced less frequently suggests that the sediment formation mechanism is the same but that water quality of the treatment effluent impacts the extent of material formation and growth of invertebrates. Overall, the results indicate that sediment formation in the distribution system is the result of complex combinations of (micro)biological and bio-chemical processes, including aggregation of particles with organic and inorganic matter, microbial growth on particles and biofilm, biomineralization, and growth of invertebrates. The determining factors to limit sediment formation, however, could not be identified. Further research is required to focus on the impact of treatment on shaping the distribution system ecosystem.

In the present research, a bio-based flame retardant (FR) was prepared using a biopolymer derived from wastewater sludge to improve the fire performance of polypropylene (PP). Extracellular polymeric substances (EPS), which were extracted from wastewater aerobic granular sludge, were absorbed into cellulose-based fibres, such as flax and toilet papers. Thermogravimetric analysis results indicated that the EPS-cellulose fibres played a significant role in enhancing the char formation of PP composite. Furthermore, the incorporation of the bio-based FR into PP restricted its vertical burning characteristics, and at the same time enhanced the tensile moduli of the composites. The reaction between phosphoric acids from EPS and hydroxyl groups of cellulose fibres improved dehydration and char formation of the composites to enhance the overall fire reaction properties. This study opens up new possibilities for the wastewater-derived biopolymer “EPS” to prepare the bio-inspired FRs for cellulose-based fibres and composites, and enhance sustainability of wastewater sludge treatment.

Polyphosphate accumulating organisms (PAOs) are responsible for enhanced biological phosphate removal (EBPR) from wastewater, where they grow embedded in a matrix of extracellular polymeric substances (EPS). EPSs comprise a mixture of biopolymers like polysaccharides or (glyco)proteins. Despite previous studies, little is known about the dynamics of EPS in mixed cultures, and their production by PAOs and potential consumption by flanking microbes. EPSs are biodegradable and have been suggested to be a substrate for other organisms in the community. Studying EPS turnover can help elucidate their biosynthesis and biodegradation cycles. We analyzed the turnover of proteins and polysaccharides in the EPS of an enrichment culture of PAOs relative to the turnover of internal proteins. An anaerobic-aerobic sequencing batch reactor (SBR) simulating EBPR conditions was operated to enrich for PAOs. After achieving a stable culture, carbon source was switched to uniformly ¹³C-labeled acetate. Samples were collected at the end of each aerobic phase. EPSs were extracted by alkaline treatment. ¹³C enrichment in proteins and sugars (after hydrolysis of polysaccharides) in the extracted EPS were measured by mass spectrometry. The average turnover rate of sugars and proteins (0.167 and 0.192 d⁻¹ respectively) was higher than the expected value based on the solid removal rate (0.132 d⁻¹), and no significant difference was observed between intracellular and extracellular proteins. This indicates that EPS from the PAO enriched community is not selectively degraded by flanking populations under stable EBPR process conditions. Instead, we observed general decay of biomass, which corresponds to a value of 0.048 d⁻¹. Key Points: • Proteins showed a higher turnover rate than carbohydrates. • Turnover of EPS was similar to the turnover of intracellular proteins. • EPS is not preferentially consumed by flanking populations.

Abstract: Pseudaminic and legionaminic acids are a subgroup of nonulosonic acids (NulOs) unique to bacterial species. There is a lack of advances in the study of these NulOs due to their complex synthesis and production. Recently, it was seen that “Candidatus Accumulibacter” can produce Pse or Leg analogues as part of its extracellular polymeric substances (EPS). In order to employ a “Ca. Accumulibacter” enrichment as production platform for bacterial sialic acids, it is necessary to determine which fractions of the EPS of “Ca. Accumulibacter” contain NulOs and how to enrich and/or isolate them. We extracted the EPS from granules enriched with “Ca. Accumulibcater” and used size-exclusion chromatography (SEC) to separate them into different molecular weight (MW) fractions. This separation resulted in two high molecular weight (> 5500 kDa) fractions dominated by polysaccharides, with a NulO content up to 4 times higher than the extracted EPS. This suggests that NulOs in “Ca. Accumulibacter” are likely located in high molecular weight polysaccharides. Additionally, it was seen that the extracted EPS and the NulO-rich fractions can bind and neutralize histones. This opens the possibility of EPS and NulO-rich fractions as potential source for sepsis treatment drugs. Key points: • NulOs in “Ca. Accumulibacter” are likely located in high MW polysaccharides • SEC allows to obtain high MW polysaccharide-rich fractions enriched with NulOs • EPS and the NulOs-rich fractions are a potential source for sepsis treatment drugs.

Eco-friendly flame retardants are greatly required to meet the expectations of low-toxicity, environmental compatibility and sustainability. Extracellular polymeric substances (EPS), the biopolymers recovered from excess granular wastewater sludge, have been successfully incorporated into poly(vinyl alcohol) (PVA) by a solution casting method. Self-extinguishment of EPS was observed in a vertical burn test. Positive effects of EPS on the reduction of heat release rate and CO emission of EPS/PVA composites were also demonstrated. The presence of various types of phosphates was detected in the EPS and a possible flame-retardant mechanism has been proposed. The investigation of using granular sludge EPS to reduce the flammability of synthetic polymers may open the possibility of converting wastewater sludge into bio phosphorus-based flame retardants.