Activated sludge (AS) wastewater treatment generates substantial excess sludge which needs to be discarded and thereby increasing operational costs. Extracellular polymeric substances (EPS) within AS present a potential resource for recovery, reducing sludge volume and mass while adding value. Achieving this goal requires a better characterization of EPS, as the relationship between its composition and the microbial communities responsible for its production remains insufficiently understood. Here, we analysed extracted EPS from 16 wastewater treatment plants across 13 countries and 5 continents and found that alkaline extractable EPS yields varied widely (2.81–18.5 wt.% VSS). The microbial community composition of abundant species varied across plants and particularly across continents and did not correlate to the EPS yield. Only sludge retention time had a significant correlation with the EPS yield (p < 0.005). Traditional colorimetric assays failed to detect compositional trends of the EPS, but Fourier Transform Infrared (FTIR) analysis indicated that extracted EPS from biological phosphorus removal systems had higher lipid and polysaccharide content, while chemical phosphorus removal systems had higher relative protein content. Thus, FTIR proved effective for distinguishing extracted EPS composition, demonstrating its potential as a high-throughput characterization tool. These findings highlighted that the wastewater treatment design and operation may shape the functional groups in EPS when using the alkaline method. More investigations are needed to find possible correlations between the composition of extracted EPS and the microbial community structure. Overall, the study presents a baseline for the amount and overall composition of biopolymers that can be extracted from global AS plants for recovery.

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.

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.

Sulfated glycosaminoglycans (sGAG) are negatively charged extracellular polymeric substances that occur in biofilms from various environments. Yet, it remains unclear whether these polymers are acquired from the external environment or produced by microbes in the biofilm. To resolve this, we analyzed the presence of sGAGs in samples of an acidophilic biofilm collected from Sulfur Cave in Puturosu Mountain (Romania), an environment that is largely inaccessible to contamination. A maximum of 55.16 ± 2.06 μg sGAG-like polymers were recovered per mg of EPS. Enzymatic treatment with chondroitinase ABC resulted in a decrease of the mass of these polymers, suggesting the structure of the recovered sGAG is similar to chondroitin. Subsequent FT-IR analysis of these polymers revealed absorbance bands at 1230 cm⁻¹, 1167 cm⁻¹ and 900 cm⁻¹, indicating a possible presence of polysaccharides and sulfate. Analysis of genomic sequences closely related to those predominant in the acidophilic biofilm, contained genes coding for sulfotransferase (an enzyme needed for the production of sGAG), which supports the hypothesis of microbial synthesis of sGAGs within the biofilm.