The vast majority of bacteria in natural and engineered environments exist in the form of biofilms, where the bacteria are embedded in a self-produced matrix of diverse biopolymers known as extracellular polymeric substances (EPS). The EPS typically consists of lipids, polysaccharides, proteins, and extracellular DNA among other components. In particular in wastewater treatment technologies, EPS plays an important role. Over the last couple of decades, a wastewater treatment technology called aerobic granular sludge (AGS) has gained increasing attention due to its more efficient performance compared to conventional activated sludge systems. In AGS, microorganisms produce a dense EPS matrix that enables granule formation and stability. Moreover, the growing emphasis on a circular economy has led to the recovery of AGS-derived EPS as a valuable biopolymer, commercially known as Kaumera..... ... Read more

Sustainable wastewater treatment system have increasingly focused on resource recovery from wastewater. Excess sludge, primarily composed of a bacterial cell matrix embedded in extracellular polymeric substances (EPS), offers significant potential in this regard. accounts for approximately 10–40% of the total dry weight of sludge and is recognized as a promising bioresource for producing valuable bioproducts. However, despite the widespread use of flocculent sludge treatment plants, the recovery potential and properties of EPS in flocculent sludge have been largely overlooked.

This thesis focuses on the extracted from flocculent sludge, with the aim of exploring their extraction potential, structural characteristics, conformations, and properties. By analyzing EPS from various full-scale and lab-scale flocculent sludge systems, it examines the factors influencing EPS extraction potential and establishes correlations between these factors and EPS formation and properties. Further investigations into EPS composition and conformation provide a deeper understanding of its structure, shedding light on its role in sludge aggregation and potential applications. This thesis bridges engineering and fundamental perspectives to advance EPS research.

Chapter 1 provides a concise introduction to the growing interest in EPS recovery, highlighting its significance and potential. It also raises key questions about EPS derived from flocculent sludge, establishing a clear roadmap for the thesis and serving as a foundation for the experimental setups in this study.
In Chapter 2, the study focuses on evaluating the EPS recovery potential from flocculent sludge. Samples were collected from various full-scale wastewater treatment plants in China, and EPS was extracted for analysis. Influent characteristics, microbial community profiles and chemical characterizations of EPS were examined to assess their correlations. The EPS yield ranged from 9% to 19% of the organic fraction of raw sludge. The findings also revealed that EPS production is highly influenced by external environmental conditions and strongly linked to bacterial diversity and abundance. This chapter highlights the significant potential of flocculent sludge for EPS recovery.

Chapter 3 aims to explore the connections between various external factors and EPS formation. Lab-scale sequencing batch reactors (SBRs) were operated under controlled conditions, with specific operational and influent parameters designed to cultivate flocculent sludge. The results revealed that sludge fed with starch-rich influent showed significantly enhanced EPS formation, while low temperatures also supported EPS synthesis. In contrast, organic loading rates and sludge retention time (SRT) had minimal impact on EPS yield. Furthermore, adaptations in EPS composition and properties indicated that both influent characteristics and operational conditions played a critical role in shaping EPS composition.
Recognizing the importance of understanding EPS structures, Chapter 4 focuses on a detailed investigation of EPS composition and structure. Extracted EPS was fractionated into distinct components for analysis. Comparisons with commercial alginates revealed that typical alginate units—guluronic acid and mannuronic acid—were absent in all EPS fractions, indicating that EPS from flocculent sludge does not contain alginate structures. Further analysis of these fractions suggested the presence of glycolipid structures, specifically highlighting the significance of lipopolysaccharides (LPS), a type of glycolipid, in EPS. This chapter not only confirmed the absence of alginates but also underscored the critical role of glycolipids in EPS composition.

Chapter 5 delves into the structure of lipopolysaccharides (LPS) and their contributions to EPS properties by comparing EPS from flocculent and granular sludge. LPS was isolated from EPS and subsequently characterized. The study found that LPS comprised approximately 25% of the organic fraction of EPS in flocculent sludge, significantly higher than the 15% observed in granular sludge. LPS from flocculent sludge exhibited unique features, including lower glycan content, shorter glycan chains, lower molecular weight, and a higher prevalence of unsaturated lipids. These structural characteristics led to inverted crosslinks in calcium-bound LPS aggregates, contributing to the fluid-like hydrogel morphology of EPS. In contrast, LPS-Ca aggregates from granular sludge exhibited a bilaminar multilayered structure, resulting in the solid, self-standing hydrogel properties of EPS.

Chapter 6 summarizes the key findings of this thesis, highlighting the insights gained into EPS recovery, structure, and properties. Additionally, it proposes ideas for future research, including exploring bacterial activities involved in EPS biosynthesis, further investigation of LPS structures and their functions, and potential applications of EPS. These suggestions aim to advance the understanding and utilization of EPS in sustainable wastewater treatment and beyond. ... Read more

Our current resource consumption practices are unsustainable due to our linear approach, which rapidly depletes resources as populations grow and demands increase. To address this issue, we are transitioning towards circular practices aimed at prolonging the use of products, ma-terials, and resources, thereby minimizing waste. This shift is critical for ensuring a sustainable and secure future for the next generations. Consider wastewater as an example: it's not merely dirty water that needs disposal; rather, it represents a concentrated source of valuable resources such as energy, reusable water, and essential nutrients like nitrogen and phosphorus. By embracing these concentrated streams, we have the opportunity to transform wastewater treatment plants into re-source recovery facilities. Here, we can efficiently extract and reuse these precious materials, thus maximizing their value and minimizing environmental impact... ... Read more

Wastewater from the food and agro-industry is filled with organic contaminants. If these substances are discharged into surface water, they promote the growth of unwanted microorganisms. To prevent this, contaminants are removed from the water through wastewater treatment. This is done using anaerobic digestion with microorganisms. Various types of microorganisms convert the organic compounds into methane gas, recovering some of the energy. The final step in anaerobic digestion, the conversion to methane, is the limiting factor in the process. A high concentration of methane-producing archaea is desired for rapid methane production.

Growing microorganisms in granules enables them to remain longer in the reactor, leading to an increased biomass concentration. The granules consist of multiple layers, each layer containing organisms that perform specific steps in the conversion to methane. These granules are a specific type of biofilm, made up of microorganisms embedded in a self-produced extracellular matrix. This matrix is composed of extracellular polymeric substances (EPS), which are produced and secreted by the microorganisms in the biofilm. EPS are a complex combination of proteins, polysaccharides, and lipids. Besides these basic polymers, combinations such as glycoproteins and lipopolysaccharides are also produced by the microorganisms. Charged polymers can form a polymer network with oppositely charged polymers or ions, contributing to the strength of the granular sludge. It is, therefore, no surprise that negatively charged particles, or acidic polymers, are often found in biofilms. However, how specific components in the EPS composition affect the structure and physical properties of granular sludge has been unclear until now.

The aim of this thesis is to study the EPS composition of anaerobic granular sludge, focusing on three main aspects: the identification of specific polymers, visualization of these polymers in the extracellular matrix, and identification of EPS synthesis pathways. While this thesis primarily characterizes the EPS composition of anaerobic granular sludge, the findings and methods are applicable to biofilms in general. By gaining a better understanding of the role of specific EPS components, we can better control biofilm processes.... ... Read more

The majority of bacteria grow in the form of microbial aggregates known as biofilms. In these biofilms, microorganisms are embedded in a mixture of extracellular polymeric substances (EPS) produced by the microorganisms themselves. EPS is a complex mixture of biopolymers of different nature, such as polysaccharides, proteins, nucleic acids or lipids, among others. In spite of the significant progress over the last decades, EPS is still a black box waiting to be opened, in terms of specific composition, function, structure and production.
Biofilms have great importance in many environmental engineering processes, as for example, aerobic granular sludge (AGS). AGS is a novel biological wastewater treatment where microorganisms are stimulated to form compact granules. Among the complex microbial community in AGS, polyphosphate accumulating organisms (PAOs) are of great importance, due to their role in phosphate removal and granule stabilization. Because of their dominance in AGS and their rapid anaerobic carbon sequestration, they are assumed to be the main EPS producer in AGS. Therefore, PAOs (specifically the well-studied “Candidatus Accumulibacter phosphatis”) can be used as model microorganism for the study of EPS of AGS.
The goal of this thesis is to study the EPS of “Ca. Accumulibacter” in terms of specific composition, application and synthesis/consumption. A better characterization of the EPS of “Ca. Accumulibacter” will lead to a comprehensive understanding of this microorganism and further optimization of the granular sludge processes, and their application...
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This thesis examines the influence of physical properties of structural EPS on granule morphology. Two types of structural EPS are found to be important. These types of structural EPS are: surface bound EPS and hydrogel forming EPS matrix. VirtualLeaf is used to model granules which contain only binding surface bound EPS. The TST model is used to find the influence of both structural EPS components. In VirtualLeaf compact granules are formed with slight bulging features, similar to granules found in literature. The TST shows that binding surface binding EPS increase the compactness of granules. Furthermore it is shown that porosity of the EPS matrix also determines compactness and maximum size of granules. The strength and measure of the EPS matrix is shown to influence granule stability. ... Read more