Melanin is a group of phenolic-quinone pigments. Natural melanin is nearly ubiquitous; found in all types of living organisms, ranging from mammals to bacteria. However, its presence and biosynthesis genomic potential in activated sludge have not been investigated. To explore this potential, melanin-like material was extracted from activated sludge collected from a municipal wastewater treatment plant. The extracted melanin-like material was characterized through biochemical analyses in comparison to synthetic melanin and humic acids that are commercially available. Metagenomic analysis of microbial community members in activated sludge and detection of tyrosine-derived melanin synthesis genes was performed. Additionally, the potential application of the extracted melanin-like material as a natural pigment was evaluated by testing its ability to color wool yarn. It was found that melanin-like material extracted from activated sludge accounted for around 11% of sludge dry mass. The isolated material displayed intrinsic autofluorescence, strong UV absorption, high oxidative stability, and free radical-rich EPR signal. FTIR analysis indicated a mixed polymer dominated by pyomelanin-like structures with eumelanin features, distinguishing it from synthetic melanin and humic acid. Metagenomic screening of the sludge community revealed widespread genomic potential for pyomelanin monomeric precursors biosynthesis across key functional genera (e.g. genera Zoogloea, Nitrotoga, Nitrosomonas, Ca. Accumulibacter, Azonexus, Ca. Competibacter, Propionivibrio, and Rhodoferax). These results suggest that microbial melanin-like material is an overlooked contributor to sludge coloration. Furthermore, the extracted pigment exhibited high affinity and wash fastness on wool fibers, demonstrating its potential for valorization as a sustainable biobased colorant.

The current research attempts to elucidate fundamental mechanistic correlation between the complex chemical architecture of wastewater-derived biopolymers – EPS (extracellular polymeric substances) and their inherent thermal properties for fire-safety applications. By integrating thermogravimetric-infrared spectroscopy with two-dimensional correlation spectroscopy, we resolve intricate mass-loss profiles into three pseudo-components (PCs), each characterised by kinetic signatures and functional group transformations. PC1 (150–350 °C, activation energy (AE) = 140–150 kJ/mol), is primarily governed by the degradation of polysaccharides and release of early-stage volatiles (H₂O, CO₂, CH₄, NH₃, and HNCO). PC2 (210–450 °C, AE = 160–175 kJ/mol), represent the transition stage dominated by proteinaceous and lipid cross-linking, which produces nitrogenous species essential for promoting condensed-phase char development. PC3 (290–600 °C, AE > 180 kJ/mol) corresponds to the decomposition of humic-like substances and subsequent aromatic condensation of stable residues. Furthermore, comparative analysis reveals that EPS extracted from activated sludge exhibits higher thermal stability and a significantly increased char yield (33.5 %) than aerobic counterpart, attributed to higher AE during the middle decomposition stage. The persistent detection of C-O-C/P–O–C and aromatic C=C vibrations up to 700 °C confirms the formation of a phosphorus-rich aromatic char structure. This multi-dimensional analytical framework moves beyond conventional TG-based pseudo-component fitting, providing high resolution interpretation of the sequential evolution of volatile species and early-stage charring mechanisms of EPS.

Extracellular polymeric substances (EPS) are essential for the structural and functional stability of aerobic granular sludge (AGS). Their complexity makes comprehensive characterization challenging and dependent on the recovery approach. This study thus investigates how alkaline extraction conditions influence the recoverability and properties of AGS-derived EPS, especially regarding structural EPS (sEPS) and related hydrogels. An advanced methodological framework was applied to compare NaOH- and Na₂CO₃-based extractions through a holistic assessment of multi-scale characterization data. The biochemical composition, molecular weight distribution and thermal behavior of EPS and/or sEPS were studied by complementary techniques (e.g. methods for protein/carbohydrate determination, size exclusion chromatography and thermogravimetry). An innovative physicochemical approach coupling small-angle X-ray scattering (SAXS) and rheology was employed to investigate the nano-scale arrangements and mechanical features of sEPS hydrogels. The NaOH-based extraction resulted in lower EPS/sEPS yields, promoting a shift towards lower molecular weight sEPS fractions. The Na₂CO₃-based method preserved larger and more thermally resistant macromolecules, suggesting milder chemical effects on the recovered EPS/sEPS. SAXS revealed that the sEPS hydrogel network consisted of 3D mass fractals and highly ordered lamellar, multilayered domains – more pronounced for Na₂CO₃-based protocol – potentially associated with lipopolysaccharide assemblies. NaOH-extracted sEPS formed stiffer hydrogels. The NaOH-based protocol likely induced harsher hydrolysis/partial degradation of specific EPS constituents (e.g. large proteins and lipidic fractions), which may not significantly contribute to, or even compromise, the hydrogel stiffness. Overall, this work demonstrates the critical role of alkaline extraction in determining EPS/sEPS yield and properties, providing valuable insights for the rational design of EPS-based biomaterials and recovery strategies.

The current study investigates the application of extracellular polymeric substances (EPS), recovered from municipal wastewater sludge, as sustainable flame-retardant materials. While EPS show significant fire-safety potential, their compositional complexity requires a clear understanding of fire performance. The primary objective of this research is to elucidate how key EPS components, such as proteins (PN), polysaccharides (PS), lipids (LP), and humic-like substances (HS), govern thermal decomposition and flammability. Thermogravimetric analysis reveals that PN and HS have higher activation energies than those of other components, enhancing EPS thermal stability. PS yield the highest char residue (48.7% at 900 °C), while PN achieve the highest graphitisation degree, highlighting their crucial roles in char formation of EPS. Pyrolysis gas analysis indicates that PN and HS are main sources of nitrogenous compounds, diluting oxygen. Moreover, flammability tests show PS have the lowest peak heat release rate (29.7 W/g), underlining the important role in increasing flame-retardancy of EPS. However, TGA-FTIR analysis indicates lipid as a primary source of combustible gases likely due to their hydrocarbon chains. This study provides valuable insight into the role of major EPS components in the flame-retardant properties and suggest a sustainable approach to enhance flame retardancy through targeted component optimisation.

This study investigates the influence of extracellular polymeric substances (EPS), recovered from wastewater sludge, on the flame-retardant and mechanical properties of wool-based fibreboards. The thermal properties of wool, resin, and EPS were analysed using thermogravimetric analysis and differential scanning calorimetry to determine manufacturing parameters and assess their impact on the thermal decomposition of the fibreboards. A specialised fibreboard manufacturing setup, incorporating a drum mixer, tube blender, and hot press, was developed to fabricate the composite boards. Results indicate that increasing the hot-pressing time enhances both flexural and internal bond strength. The incorporation of EPS significantly improves the internal bond strength compared to fibreboards without the biopolymer. Moreover, the combined effects of wool and EPS promote effective char formation and lead to a V-0 rating, showing self-extinguishing behaviour in vertical burn tests. Cone calorimeter analysis reveals that while EPS contributes to a reduction in the heat release rate, its effect reaches a saturation point. However, the fire growth index, along with barrier and protective effect values, demonstrates that EPS effectively mitigates fire spread and propagation. These findings highlight the potential of wastewater-derived EPS as a sustainable additive for enhancing the fire resistance and mechanical integrity of wool-based fibreboards.

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.

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.

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.

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.