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.]

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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. @en

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.

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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.

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Sialic acids are a family of nine-carbon negatively charged carbohydrates. In animals, they are abundant on mucosa surfaces as terminal carbohydrates of mucin glycoproteins. Some commensal and pathogenic bacteria are able to release, take up and catabolize sialic acids. Recently, sialic acids have been discovered to be widespread among most microorganisms. Although the catabolism of sialic acids has been intensively investigated in the field of host-microbe interactions, very limited information is available on microbial degradation of sialic acids produced by environmental microorganisms. In this study, the catabolic pathways of sialic acids within a microbial community dominated by 'Candidatus Accumulibacter' were evaluated. Protein alignment tools were used to detect the presence of the different proteins involved in the utilization of sialic acids in the flanking populations detected by 16S rRNA gene amplicon sequencing. The results showed the ability of Clostridium to release sialic acids from the glycan chains by the action of a sialidase. Clostridium and Chryseobacterium can take up free sialic acids and utilize them as nutrient. Interestingly, these results display similarities with the catabolism of sialic acids by the gut microbiota. This study points at the importance of sialic acids in environmental communities in the absence of eukaryotic hosts.

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