Structural extracellular polymeric substances (structural EPS) can form stable hydrogels and are considered to be responsible for the stability of biofilms and aerobic granular sludge. Structural EPS were extracted from aerobic granular sludge and characterized for their gel-forming capacity with different alkaline earth and transition metal ions. The structural EPS hydrogels were compared to alginate hydrogels. Alginate is a well characterized polymer which is able to form stiff hydrogels with multivalent ions. The stiffness of the obtained hydrogels was measured with dynamic mechanical analysis and quantified by the Young's modulus. Furthermore the stability of structural EPS hydrogels towards disintegration in the presence of ethylenediaminetetraacetic acid (EDTA) was evaluated at pH 4.5–10.5 and compared to that of alginate, polygalacturonic acid and κ-carrageenan. The stiffness of alginate hydrogels was multiple times higher than that of structural EPS. Alkaline earth metals resulted in stiffer alginate hydrogels than transition metals. For structural EPS this trend was opposite to alginate. Independent of the pH, polysaccharide hydrogels were quickly disintegrated when being exposed to EDTA. Structural EPS hydrogels demonstrated greater stability towards EDTA and were still intact after one month at pH 4.5–8.5. It is suggested that the gelling mechanism of structural EPS is not only related to metal ion complexation of the polymers, but to a combination of interactions of multiple functional groups present in structural EPS. This study helps to further understand and characterize structural EPS from aerobic granular sludge, and therewith understand its stability and that of biofilms in general.

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Glycosaminoglycans (GAGs) are linear heteropolysaccharides containing a derivative of an amino sugar. The possibility of the presence of GAGs in aerobic granular sludge was studied by combining SDS-PAGE with Alcian Blue staining (at pH 2.5 and 1), FTIR, mammalian Hyaluronic acid and sulfated GAG analysis kits, enzymatic digestion and specific in situ visualization by Heparin Red and lectin staining. GAGs, including Hyaluronic acid-like and sulfated GAGs-like polymers were found in aerobic granular sludge. The sulfated GAGs-like polymers contained Chondroitin sulfate and Heparan sulfate/Heparin based on their sensitivity to the digestion by Chondroitinase ABC and Heparinase I & III. Heparin Red and lectin staining demonstrated that, the sulfated GAGs-like polymers were not only present in the extracellular matrix, but also filled in the space between the cells inside the microcolonies. The GAGs-like polymers in aerobic granules were different from those produced by pathogenic bacteria but resemble those produced by vertebrates. Findings reported here and in previous studies on granular sludge described in literature indicate that GAGs-like polymers might be widespread in granular sludge/biofilm and contribute to the stability of these systems. The extracellular polymeric substances (EPS) in granular sludge/biofilm are far more complicated than they are currently appreciated. Integrated and multidisciplinary analyses are significantly required to study the EPS.

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Aerobic granular sludge (AGS) technology is an alternative to conventional activated sludge to reduce the process footprint and energy consumption. Strategies for the efficient management of its produced biomass, that is grown in a granular morphology as well, need further development. Anaerobic digestion (AD) is commonly applied in waste activated sludge (WAS) treatment and is a potential option also for produced AGS treatment. In earlier studies, the biochemical methane potential of AGS was found lower than that of WAS both grown in full-scale municipal wastewater treatment systems. In order to understand this difference, this study aimed to investigate the anaerobic conversion of structural extracellular polymeric substances (SEPS), which is a type of gel-forming biopolymer, being responsible for the aggregation of sludge. Using WAS and AGS as substrates, a comparative AD batch experiment was performed for 44 days during which the SEPS fraction was extracted from both types of sludge. The changes in the SEPS chemical composition was analysed by Fourier transformed infrared spectroscopy and three-dimensional excitation and emission matrix analysis. In addition, the mechanical strength of hydrogels of extracted polymers cross-linked with Ca2+ ions was investigated by dynamic mechanical analysis. Results showed that the amount of SEPS was reduced by 26% in AGS (SEPSAGS) and by 41% in WAS (SEPSWAS), respectively. Polysaccharides and, to a lesser extent, the proteins in the SEPSAGS were more refractory compared to those in SEPSWAS. This resulted in a lower loss of the gel stiffness of SEPSAGS than that of SEPSWAS during the AD process. Moreover, the release of SEPS from tightly bound EPS to loosely bound EPS were observed in both types of sludge, but that in AGS exhibited a lower transition rate. The observed properties explain the distinct differences in anaerobic biodegradability, the slower decomposition of the sludge structure, as well as the better dewaterability of AGS as compared to WAS after the AD process.@en

Biofilms are pervasive in hydrated environments including wastewater and drinking water systems. A novel promising biological wastewater treatment process offering several advantages towards wastewater treatment with the conventional activated sludge process is the aerobic granular sludge process. Aerobic granular sludge is a special kind of biofilm of spherical shape formed by microorganisms without the addition of carrier material. Biofilms are microbial aggregates composed of microorganisms and extracellular polymeric substances (EPS). EPS are a complex mixture of proteins, polysaccharides, uronic acids, nucleic acids lipids and humic substances. EPS have multiple important functions within a biofilm. They contribute to the initial aggregation of microbial cells and form a highly hydrated matrix being responsible for the structural integrity of a biofilm. By this EPS also provide protection, can serve as a nutrient source and bind extracellular enzymes. Being a complex mixture of multiple compounds makes EPS analysis challenging and therefore the actual composition and structure of the matrix of biofilms is still largely unknown. Aerobic granular sludge and part of its EPS, structural EPS, has hydrogel properties. These structural EPS can be extracted from the granules and were shown to be strongly linked to the structural integrity of the sludge. Characterization of the structural EPS will help to understand the stability of granular sludge and in general of biofilms. The focus of this thesis was to analyze the composition of structural EPS from aerobic granular sludge and to analyze its hydrogel characteristics. Additionally challenges and shortcomings concerning EPS extraction and characterization are illustrated and discussed. Chapter 1 gives a general introduction into biofilms and their EPS, as well as EPS extraction. Issues with current EPS characterization are provided and the outline of this thesis is presented. In Chapter 2 the impact of the extraction method on aerobic granular sludge and the obtained EPS was demonstrated with six different EPS extraction methods including mechanical and chemical treatment. Results showed that to obtain structural EPS it is necessary to dissolve the granular matrix. To dissolve the granular matrix harsh extraction methods are required, and there is no ”one fits all” method to dissolve the granular matrix for structural EPS extraction. Chapter 3 illustrates and discusses shortcomings of current EPS analysis with colorimetric methods for the quantification of proteins, sugars, uronic acids and humic substances. Drawbacks of these colorimetric methods include: a high dependency on the standard compound selection, a lack of suitable standards which feature a similar composition with the analyzed sample and cross-interference among EPS compounds in the measurements. Results showed that, these methods are not suitable to accurately analyze complex samples. The complexity of structural EPS was illustrated by the overall composition of granular sludge structural EPS: besides a protein fraction, the carbohydrate part itself contained a sugar alcohol, seven neutral sugars, two amino sugars and two uronic acids. Simply depending on colorimetric methods for EPS analysis is not recommended. Novel analytic methods need to be developed and implemented for in depth biofilm EPS analysis. In Chapter 4 structural EPS hydrogels formed with metal ions were characterized in terms of gel stiffness and structural homogeneity. Additionally the influence of the metal ion chelating reagent EDTA on the structural integrity of ionic structural EPS hydrogels was investigated. For comparison, alginate, polygalacturonic acid and κ- carrageenan were used as a reference material. The structural EPS hydrogels were less stiff than alginate hydrogels. The structure of lyophilized ionic structural EPS hydrogels was visualized with environmental SEM. Different metal ions had a different impact on the structure of the lyophilized gels. In comparison to alginate, polygalacturonic acid and κ-carrageenan, the integrity of structural EPS hydrogels was less sensitive to EDTA. After one month incubation in an EDTA solution, structural EPS gel beads were still present as a gel while the reference polysaccharide hydrogels failed to keep the gel structure. Apparently structural EPS have a different ionic hydrogel formation mechanism. Multiple functional groups are suggested to be involved in the gel formation of structural EPS. Chapter 5 focused on the analysis of strongly anionic macromolecules in the EPS of aerobic granular sludge. The presence of glycosaminoglycans was evaluated by SDSPAGE analysis, hyaluronic acid and sulfated glycosaminoglycan quantification kits for the mammalian extracellular matrix and glycosaminoglycan specific enzymatic digestion. The linking between sulfated glycosaminoglycans and proteins was analyzed by proteolytic enzymatic digestion. Furthermore, Heparin Red staining was used to visualize the distribution of the anionic macromolecules in the granular matrix. Macromolecules similar to Hyaluronic acid and sulfated glycosaminoglycans were discovered in the EPS, hence named Hyaluronic acid-like and sulfated glycosaminoglycans-like compounds. Sulfated glycosaminoglycans-like compounds were bound to proteins. In aerobic granular sludge the strongly anionic molecules were distributed in the microcolonies, at the outer part of the microcolonies and within the extracellular matrix between the colonies. Glycosaminoglycans-like compounds showed to be comparable to those of vertebrates. Structural EPS were therefore much more complicated than expected. Chapter 6 represents the outlook of this thesis. Results from the previous chapters are summarized and suggestions for future EPS research are given. Suggestions include extraction of EPS, chemical analysis of EPS and general approaches. @en

Sialic acids have been discovered in the extracellular polymeric substances (EPS) of seawater-adapted aerobic granular sludge (AGS). Sialic acids are a group of monosaccharides with a nine-carbon backbone, commonly found in mammalian cells and pathogenic bacteria, and frequently described to protect EPS molecules and cells from attack by proteases or glycosidases. In order to further understand the role of these compounds in AGS, lectin staining, genome analysis of the dominant bacterial species, and shielding tests were done. Fluorescence lectin bar-coding (FLBC) analysis showed an overlap with protein staining, indicating presence of sialoglycoproteins in the EPS matrix. Genome analysis gives a positive indication for putative production of sialic acids by the dominant bacteria Candidatus Accumulibacter. FT-IR analysis shows upon selective removal of sialic acids a decrease in carbohydrates, extension of the protein side chain, and exposure of penultimate sugars. Enzymatic removal of sialic acids results in the removal of galactose residues from the EPS upon subsequent treatment with β-galactosidase, indicating a linkage between galactose and sialic acid at the terminus of glycan chains. This work indicates the importance of sialic acids in the protection of penultimate sugar residues of glycoproteins in EPS, and provides basis for future research in the composition of EPS from biofilms and granular sludge.

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Biofilm structure and functionality depend on extracellular polymeric substances (EPS), but analytical methods for EPS often lack specificity which limits progress of biofilm research. EPS were extracted from aerobic granular sludge and analyzed with frequently applied colorimetric methods. The colorimetric methods were evaluated based on their applicability for EPS analysis. EPS fractions of interest were proteins, sugars, uronic acids and phenolic compounds. The applied methods (Lowry method, bicinchoninic acid assay, phenol sulfuric acid method, carbazole sulfuric acid method) were investigated in terms of their sensitivity towards the selected standard compound. Interference of compounds present in EPS with the colorimetric methods was further evaluated. All methods showed to be highly depending on the choice of standard compound and susceptible towards interference by compounds present in EPS. This study shows that currently used colorimetric methods are not capable of accurately characterizing EPS. More advanced methods are needed to be able to draw conclusions about biofilm composition, structure and functionality.

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To evaluate and develop methodologies for the extraction of gel-forming extracellular polymeric substances (EPS), EPS from aerobic granular sludge (AGS) was extracted using six different methods (centrifugation, sonication, ethylenediaminetetraacetic acid (EDTA), formamide with sodium hydroxide (NaOH), formaldehyde with NaOH and sodium carbonate (Na₂CO₃) with heat and constant mixing). AGS was collected from a pilot wastewater treatment reactor. The ionic gel-forming property of the extracted EPS of the six different extraction methods was tested with calcium ions (Ca²⁺). From the six extraction methods used, only the Na₂CO₃ extraction could solubilize the hydrogel matrix of AGS. The alginatelike extracellular polymers (ALE) recovered with this method formed ionic gel beads with Ca²⁺. The Ca²⁺-ALE beads were stable in EDTA, formamide with NaOH and formaldehyde with NaOH, indicating that ALE are one part of the structural polymers in EPS. It is recommended to use an extraction method that combines physical and chemical treatment to solubilize AGS and extract structural EPS.

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This study focused on characterizing the structure of biofilms developed on carriers used in lab-scale moving bed biofilm reactors. Both light microscopy (2D) and optical coherence tomography (OCT) were employed to track the biofilm development on carriers of different geometry and under different aeration rates. Biofilm structure was further characterized with respect to average biofilm thickness, biofilm growth velocity, biomass volume, compartment filling degree, surface area, etc. The results showed that carriers with a smaller compartment size stimulated a quick establishment of biofilms. Low aeration rates favored fast development of biofilms. Comparison between the results derived from 2D and 3D images revealed comparable results with respect to average biofilm thickness and compartment filling degree before the carrier compartments were fully willed with biomass. However, 3D imaging with OCT was capable of visualizing and quantifying the heterogeneous structure of biofilms, which cannot be achieved using 2D imaging.

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