The long-term effects of environmental conditions, such as seawater salinity, on the extracellular investigated EPS changes during a stepwise increase in salinity (0–4%), renewing over 90% of biomass at each condition. Stable granulation, complete anaerobic acetate uptake, and phosphate removal were maintained throughout. FT-IR of granules showed significant changes in glycans (1025 cm⁻¹) and sialic acid (1730 cm⁻¹), which were reflected in the EPS. Lectin microarray revealed that increasing salinity reduced glycan diversity in EPS glycoproteins, while increasing negatively charged groups, including sialic acids and sulfated groups. At 4% salinity, EPS negative charge increased by 19.8% compared to 0%. Microbial community composition shifted from a diverse mix (Dechloromonas; 23%, “Candidatus Competibacter”; 13%, “Candidatus Accumulibacter”; 28%) at 0% to a dominant (69% – 75%) unclassified Accumulibacter clade I species at 1 - 4% salinity. Metaproteomic analysis showed strong upregulation of genes of “Ca. Accumulibacter” involved in monosaccharide, lipopolysaccharide, and peptidoglycan biosynthesis from 3% - 4% salinity, indicating its adaptation to salinity stress. Dechloromonas and “Ca. Competibacter” represented a minor or a non-significant fraction of those proteins related to glycan synthesis across the salinities. Despite that no glycoprotein biosynthesis pathways were identified in the metaproteomic data, three putative glycoproteins produced by “Ca. Accumulibacter” were detected across all conditions. They were downregulated as the salinity increased. These findings highlight how “Ca.Accumulibacter” dynamically adapts its EPS, particularly glycoprotein glycans, in response to increasing salinity, offering new insights into EPS adaptation under environmental stress. ... Read more

The authors would like to inform readers that a sentence was inadvertently omitted from the ‘Abstract’ section during the production process. The correct ‘Abstract’ section should read as follows: The long-term effects of environmental conditions, such as seawater salinity, on the extracellular polymeric substances (EPS) of aerobic granular sludge (AGS) remain poorly understood. This study investigated EPS changes during a stepwise increase in salinity (0–4%), renewing over 90% of biomass at each condition. Stable granulation, complete anaerobic acetate uptake, and phosphate removal were maintained throughout. FT-IR of granules showed significant changes in glycans (1025 cm⁻¹) and sialic acid (1730 cm⁻¹), which were reflected in the EPS. Lectin microarray revealed that increasing salinity reduced glycan diversity in EPS glycoproteins, while increasing negatively charged groups, including sialic acids and sulfated groups. At 4% salinity, EPS negative charge increased by 19.8% compared to 0%. Microbial community composition shifted from a diverse mix (Dechloromonas; 23%, “Candidatus Competibacter”; 13%, “Candidatus Accumulibacter”; 28%) at 0% to a dominant (69% – 75%) unclassified Accumulibacter clade I species at 1 - 4% salinity. Metaproteomic analysis showed strong upregulation of genes of “Ca. Accumulibacter” involved in monosaccharide, lipopolysaccharide, and peptidoglycan biosynthesis from 3% - 4% salinity, indicating its adaptation to salinity stress. Dechloromonas and “Ca. Competibacter” represented a minor or a non-significant fraction of those proteins related to glycan synthesis across the salinities. Despite that no glycoprotein biosynthesis pathways were identified in the metaproteomic data, three putative glycoproteins produced by “Ca. Accumulibacter” were detected across all conditions. They were downregulated as the salinity increased. These findings highlight how “Ca. Accumulibacter” dynamically adapts its EPS, particularly glycoprotein glycans, in response to increasing salinity, offering new insights into EPS adaptation under environmental stress. ... Read more

In this research, a method has been developed to measure the charge density of complex biopolymer systems. Extracellular polymeric substances (EPS), obtained from wastewater treatment plants (WWTPs), are such a complex mixture of biopolymers and have shown to be difficult to characterize. One important characteristic for the development of applications such as green batteries, heavy metal sorption, and use as a bio flocculant is the charge density. Alginate was used as a model compound because of its well-characterized structure and known charge density. The method was optimized by varying the sample and titrant concentrations, and the titration speed. A sample concentration of 0.01 M was shown to provide the most accurate results, and the titrant concentration and titration speed had less effect on the measured charge density. With the optimal measurement settings, the method was validated and used to determine differences in charge density among EPS extracts from various wastewater treatment sludges. The different EPS samples have varying charge densities between 1.18 and 3.57 mmol/g, and the first derivative of the pH during titrations showed significant differences between samples. The resulting protocol provides a robust, reproducible, and precise approach for analyzing the EPS charge density. Furthermore, it expands the potential of conductometric methods, as systematic differences in the pH derivative curves were observed. ... Read more

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. ... Read more