Structural Extracellular Polymeric Substances from Aerobic Granular Sludge

Simon Felz

Research output: ThesisDissertation (TU Delft)

316 Downloads (Pure)


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.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Delft University of Technology
  • van Loosdrecht, Mark C.M., Supervisor
  • Lin, Y., Advisor
Award date9 Dec 2019
Print ISBNs978-94-028-1775-1
Publication statusPublished - 2019


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