Industrial wastewaters often have unique properties and contain impurities that pose a significant challenge to their treatment. Lab-scale experiments were performed to provide answers on the feasibility of aerobic granular sludge (AGS) technology for the treatment of organically polluted industrial wastewater.
Glycerol is found in a variety of industrial effluents such as biodiesel and epoxy resin production facilities. However, little is known about the conversion and the impact of glycerol on AGS processes. Chapter 2 describes glycerol conversion in AGS capable of enhanced biological phosphorus removal (EBPR). Robust granules with good phosphorus removal capabilities were formed in an AGS sequencing batch reactor fed with glycerol as the sole carbon source. The interaction between the fermentative conversion of glycerol and product uptake by polyphosphate accumulating organisms (PAO) was studied using stoichiometric and microbial community analysis. The analysis of the biomass identified a community dominated by Actinobacteria (Tessaracoccus and Micropruina) and a typical PAO known as Ca. Accumulibacter. Glycerol uptake facilitator (glpF) and glycerol kinase (glpK), two proteins involved in the transport of glycerol into the cellular metabolism, were only observed in the genome of the Actinobacteria. The anaerobic conversion appeared to be a combination of substrate fermentation and product uptake-type reaction. Initially, glycerol fermentation led mainly to the production of 1,3-propanediol (1,3-PDO) which was not taken up under anaerobic conditions. Despite the aerobic conversion of 1,3-PDO, stable granulation was observed. Over time, 1,3-PDO production decreased and complete anaerobic COD uptake was observed. Overall, the results demonstrate that glycerol-containing effluents can effectively be treated by the AGS process via a collaboration between fermentative and polyphosphate accumulating organisms.
The sugar production industries generate a significant amount of wastewater rich in sugars such as glucose. In Chapter 3, glucose conversion by AGS and its impact on phosphate removal is studied. Long-term stable phosphate removal and successful granulation were observed. Glucose was rapidly taken up with a rate of 273 mg/gVSS/h at the start of the anaerobic phase, while phosphate was released during the full anaerobic phase. Lactate was produced as the main product during glucose consumption, which was anaerobically consumed once glucose was depleted. Other products such as propionate, acetate, and formate were also detected in minor quantities. The phosphate release appeared to be directly proportional to the uptake of lactate. The ratio of phosphorus released to glucose carbon taken up over the full anaerobic phase was 0.25 Pmol/Cmol. Along with glucose and lactate uptake in the anaerobic phase, polyhydroxyalkanoates and glycogen storage were observed. Quantitative fluorescence in-situ hybridization (qFISH) revealed that PAOs accounted for the majority of the total biovolume. Anaerobic conversions were evaluated based on theoretical ATP balances to provide the substrate distribution among the dominant genera. In conclusion, this research shows that AGS can be applied for the treatment of glucose-containing effluents and it is a suitable substrate for achieving phosphate removal.
Industrial wastewaters often have high levels of salt, either due to seawater or e.g. sodium chloride (NaCl) usage in the processing. In Chapter 4, the impact of NaCl concentration gradient and seawater on the granulation and conversion processes of AGS was investigated. Glycerol was used as the carbon source since it is regularly present in industrial wastewaters, and to allow the evaluation of microbial interactions that reflect industrial effluents. Smooth and stable granules as well as EBPR were achieved up to 20 g/L NaCl or when using seawater. However, at NaCl levels comparable to seawater strength (30 g/L) incomplete anaerobic glycerol uptake and aerobic phosphate uptake were observed, the effluent turbidity increased, and filamentous granules began to appear. The latter was likely due to the direct aerobic growth on the leftover substrate after the anaerobic feeding period. In all reactor conditions, except the reactor with 30 g/L NaCl, Ca. Accumulibacter was the dominant microorganism. In the reactor with 30 g/L NaCl, an increase in the genus Zoogloea was observed. Throughout all reactor conditions, Tessaracoccus and Micropruina, both actinobacteria, were present which were likely responsible for the anaerobic conversion of glycerol into volatile fatty acids. None of the glycerol metabolizing proteins were detected in Ca. Accumulibacter which supports previous findings that glycerol can not be directly utilized by Ca. Accumulibacter. The exposure of salt-adapted biomass to hypo-osmotic conditions led to significant trehalose and PO43--P release which can be related to the osmoregulation of the cells. The findings provide insights into the effect of salt on the operation and stability of the AGS processes and suggest that maintaining a balanced cation ratio is likely to be more important for the operational stability of the system than absolute salt concentrations.
Extracellular polymeric substances (EPS) are important constituents of biofilms with promising application potential. The properties of EPS vary depending on environmental conditions and microbial communities which also entails inconsistencies in the material. In Chapter 5, we investigated the EPS of AGS grown under varying salinities induced by NaCl concentration gradient and seawater conditions. Fourier transform infrared (FTIR) spectroscopy revealed the likely presence of polysaccharides, phosphates, proteins, carboxylic esters, and lipids in all extracted EPS. Further analysis with 2-D correlation spectroscopy identified notable differences in various regions corresponding particularly to phosphate and glycan functional groups. Sugar monomer analysis of acid-hydrolysed EPS identified eight monosaccharides, with glucose dominant in saltwater conditions and glucosamine in freshwater. We further evaluated the potential of the extracted EPS as a bio-based flame retardant, via burning tests on EPS-coated cellulose fibres. The tests indicated a linear correlation between increased residual mass and the condensed phosphate content in the EPS, suggesting that higher condensed phosphate levels enhance the flame-retardant properties of the EPS. The EPS from saline conditions had higher condensed phosphate content in contrast to the freshwater EPS with higher orthophosphate fraction. In conclusion, the findings highlighted the potential of wastewater-derived EPS as a bio-based flame retardant and the impact of salt on EPS properties.
Finally, the thesis is concluded with Chapter 6 providing an outlook on the future research, economics, and application of AGS technology. Overall, the findings suggest that AGS technology can be applied for the treatment of industrial wastewater containing salts (pure NaCl or sea salt crystals) as well as glycerol and glucose as organic pollutants, with the added benefit of recovering valuable resources.@en

Biology is full of complexities, and the more we learn, the more we realize how much remains unknown. A major debate in microbiology is whether DNA alone dictates an organism’s function or if metabolism and energy flows play an equally fundamental role. This question is particularly relevant for microbes in dynamic environments, where survival depends on metabolic adaptability.

This thesis focuses on “Candidatus Accumulibacter”, a key microorganism in wastewater treatment that removes excess phosphorus from water. These bacteria endure feast-famine cycles by storing and utilizing energy reserves as conditions change. While extensively studied, much remains unknown about their metabolic strategies and how environmental factors shape their function. This research combines computational models, laboratory cultivation, and multi-omics analysis to explore how “Ca. Accumulibacter” optimizes its metabolism.

Chapter 1 introduces the central debate: Is DNA the sole blueprint for microbial function, or do metabolism and energy constraints shape microbial behavior? It traces the shift from biochemical models to genome-centric approaches and highlights the potential of a metabolism-first perspective. It also contextualizes “Ca. Accumulibacter” within existing research, outlining its role in biological phosphorus removal and summarizing past findings.

Chapter 2 investigates extracellular polymeric substances (EPS) produced by “Ca. Accumulibacter”, revealing novel glycans and glycoproteins that challenge genome-based predictions. These biomolecules are crucial for biofilm formation and microbial interactions, emphasizing the need for direct biochemical analysis alongside genetic data.

Chapter 3 uses elementary flux mode analysis (EFMA) to map the metabolic potential of “Ca. Accumulibacter”. While genome annotations suggest flexibility, thermodynamic constraints limit feasible metabolic strategies, highlighting the role of energy availability in shaping microbial function.

Chapter 4 introduces the development of the Conditional Flux Balance Analysis (cFBA) Toolbox, an open-source Python framework for modeling metabolism in fluctuating environments. Unlike conventional models that assume steady-state conditions, cFBA enables dynamic predictions of resource allocation over time.

Chapter 5 explores the impact of temperature on “Ca. Accumulibacter” metabolism using cFBA. The findings confirm that biomass synthesis is mainly aerobic but also uncover metabolic shifts at lower temperatures that influence phosphorus removal efficiency and microbial competition.

Chapter 6 examines how “Ca. Accumulibacter” metabolizes multiple substrates simultaneously, revealing unexpected synergies that enhance survival in microbial communities. Combining experimental enrichment cultures with cFBA, this study identifies key metabolic trade-offs and resource optimization strategies.

Finally, Chapter 7 synthesizes the thesis findings, advocating for a shift beyond genome-based interpretations toward a metabolism-centric understanding of microbial function. It discusses broader implications for microbial ecology, wastewater engineering, and metabolic modeling, emphasizing the need for multi-omics approaches and potential applications in synthetic biology.
By integrating experimental and computational approaches, this research deepens our understanding of how “Ca. Accumulibacter” thrives in fluctuating environments. More broadly, it highlights the importance of metabolism and energy flows in shaping microbial function, offering insights that extend beyond wastewater treatment to microbial ecology and engineered bioprocesses.@en

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...@en

Microbial communities drive the nitrogen cycle, a fundamental process sustaining life on Earth. However, human activities have disrupted this balance, leading to excessive emissions of nitrous oxide (N2O), a potent greenhouse gas with nearly 300 times the global warming potential of carbon dioxide and a significant contributor to ozone layer depletion. Despite the urgency to reduce emissions, they are projected to increase by 50% in the next 50 years. Our ability to mitigate these emissions is limited by our incomplete understanding of the microbial complexity driving them. To develop effective mitigation strategies, we must determine how microbial communities regulate nitrogen transformations across diverse environments, from natural ecosystems such as soils and oceans to managed and engineered systems like agricultural soils and wastewater treatment plants (WWTPs).

By integrating genomic, proteomic, and metabolic insights, this thesis explores the complexity of microbial nitrogen cycling and N2O emissions.@en

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. @en

Desalination plays a vital role in addressing water scarcity, but its high energy consumption and brine, a saline waste stream, disposal pose significant environmental and economic challenges. Seawater is a rich source of valuable and scarce materials that are lost when brine is discharged, making resource recovery a promising approach to improve sustainability. Integrating multiple technologies to recover water and valuable materials improves technological performance, but it introduces technical, economic, and societal complexities.
While desalination with resource recovery offers an alternative source of water, salts, and chemicals, its sustainability depends on local conditions and necessitates a holistic evaluation. Assessing these systems is particularly complex when water, a primary good, is among the recovered products. This research aims to refine assessment methodologies and explore trade-offs in integrated desalination and brine treatment. It adopts an exploratory, mixed-methods approach, beginning with a systematic literature review and the development of a sustainability assessment framework that prioritizes stakeholder participation.
In Chapter 2, the current sustainability assessment frameworks in desalination, water treatment, and resource recovery were reviewed and analysed. The literature review identified critical shortcomings in current sustainability assessments for seawater desalination and brine treatment systems. These assessments notably neglect social aspects and stakeholder involvement. To address these deficiencies, we proposed a new Sustainability Assessment (SA) framework that integrates participatory multi-criteria analysis and value-sensitive design into the decision-making process.
An open-source software tool in Python was developed in Chapter 3 to simulate the desalination and mineral recovery processes, providing data that will inform later assessments. The outputs from this software directly support the analyses presented in Chapters 4–7, illustrating its integral role in this thesis and its potential for broader applicability.
The value-sensitive design (VSD) approach was applied in Chapter 4 to design and evaluate integrated seawater desalination and brine treatment, ensuring that technical scenarios align with societal values. Four configurations were assessed for trade-offs between resource recovery, energy consumption, and environmental impact. While maximizing water and salt recovery improves resource security, it increases energy use and CO₂ emissions. The study highlights the need for region-specific solutions and demonstrates how VSD fosters stakeholder dialogue, supporting sustainable and socially acceptable designs. These scenarios serve as the basis for analysis in subsequent chapters.
In Chapter 5, the economic performance of desalination systems focused on resource recovery was assessed using the levelized cost indicator. Allocation factors were used to fairly distribute costs and income from recovered products. A comparison of traditional Non-allocation and novel cost calculation methods revealed that the Non-allocation method overestimates production costs, resulting in inflated product prices. The Economic allocation approach, by redistributing costs to higher-value products, assigns a minimal percentage to water costs, unlike the heavy loading seen with Non-allocation.
Chapter 6 investigates the environmental performance of integrated desalination and brine treatment systems for resource recovery using Life Cycle Assessment (LCA). The study highlights how key methodological choices—like functional unit and treatment of waste heat—substantially affect results. Overall, resource recovery systems demonstrated superior performance compared to conventional production systems of the same product basket, highlighting the need for integrated practices.
Finally, the effect of interdependence among decision criteria in the multi-criteria decision-making process for sustainability assessment was evaluated in Chapter 7. By combining the Best-Worst Model and the Decision-Making Trial and Evaluation Laboratory technique, we proposed a novel weighting method that accounts for interdependencies. Applied to desalination and brine treatment, results showed that while numerical impacts are moderate, capturing interdependencies improves conceptual understanding—particularly in single-stakeholder settings.
In Chapter 8, a summary of the main findings of this thesis is provided, along with the limitations of this work and an outlook for future research directions based on these findings.
@en

Drinking water production works well, but we can do better.

Anaerobic groundwater is an excellent drinking water source. It presents several advantages over its counterpart, surface water, such as constant quality and temperature, and it is considered to be microbiologically safe. The main contamination sources of anaerobic groundwater are the decomposition of natural organic matter and the dissolution of soil minerals. The first one produces compounds such as ammonia, while the second introduces manganese, iron, and trace metals. Iron, ammonia and manganese must be removed from groundwater to produce drinking water. For this purpose, humans have been using rapid sand filtration - preceded by an aeration step - for over a century.

The purpose of aeration is to strip out undesired gases and to introduce oxygen up to saturation levels. At this oxidation-reduction potential, iron, ammonia and manganese are oxidized by different physical-chemical and biological processes in the subsequent rapid sand filter. As a result, most contaminants precipitate, forming solids that are captured by the filter, and clean water is produced. Although widely used and robust, solid understanding of the intricacies of rapid sand filters is still missing. A high degree of complexity is hidden behind their seemingly simple working principles.

The main reason underneath this extraordinary complexity is the high oxygen load introduced during the aeration step. The saturation of anaerobic groundwater with oxygen onsets a series of simultaneous, interwoven and uncontrolled reactions whose nature and contribution to the overall process remains unknown and generally unpredictable. This process convolution precludes understanding and optimization of rapid sand filtration.

The overarching goal of this thesis is to gain knowledge to advance towards the design of high-flow, resource-efficient sand filters. To do so, we must be able to understand the mechanisms that govern which reactions take place, in which order they occur, and how they affect each other, which will ultimately allow us to predict and control i) microbial community assembly and performance and ii) the interplay between chemical and biological reactions. In the first part of this thesis, we focused on gaining mechanistic understanding of how current sand filters work using laboratory, pilot, and full-scale experiments. In the second one, we used this freshly acquired knowledge to design and test novel systems…
@en

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....@en

Organic waste constitutes a substantial portion of global waste, posing environmental challenges. Anaerobic Digestion (AD) plants offer a circular bioeconomy solution by converting organic waste into renewable energy and nutrients. While AD primarily yields methane, the resulting nutrient-rich digestate holds significant agronomic value for land applications. However, the direct application of digestate is banned in several countries due to environmental and health hazards, including the presence of contaminants of emerging concern (CECs). This circumstance leads to landfilling as the primary alternative—a non-sustainable solution.

This thesis explores the application of ozone-based Advanced Oxidation Processes (AOPs) as a potential post-AD treatment for liquid digestate, aligning with the fundamental objectives of fostering sustainable circular economy practices in waste management.....@en

Alkaliphiles thrive in environments with a pH of 8.5 or above, while maintaining an internal pH closer to neutral. Thus, alkaliphilic microorganisms have a proton gradient inverted with respect to the normal orientation. Intuitively, this would nullify the potential to generate energy via respiration with regularly oriented respiratory chains that rely on proton-coupled ATP synthases. Yet, alkaliphilic respiratory chains are oriented traditionally and are actively used. The question therefore is how they are able to create conditions conducive to such behaviour. In addition, attempts to answer that question will hopefully also clarify how alkaliphiles acidify their cytoplasm with respect to the exterior milieu in the first place. This thesis details methods required to study these questions and provides some answers regarding alkaliphilic life. This thesis focuses on a single category of alkaliphiles: the low-salt gram positive alkaliphiles. These microbes have just a single membrane, the proteins therein, and a cell wall to generate conditions suitable for energy generation and other transport mechanisms. In short, it can be regarded as the most basic system to study an alkaline, or basic, problem....@en

There is a rising concern on the sustainability of Zero Liquid Discharge (ZLD) desalination. In this study, Multi-criteria Decision-making (MCDM) is applied to compare the sustainability performance of various ZLD scenarios, which is part of a Strong Sustainability (SS) assessment of desalination. ZLD desalination systems are considered to be complex and have intricate interdependence among various factors, which were seldom recognized in previous research. The purpose of this study is to explore the impact of interdependence among criteria on the MCDM process, with a desalination case study from Water Mining project used for demonstration. A transparent MCDM methodology is proposed in this study, which consists of Best Worst Method – Decision Making Trial and Evaluation Laboratory (BWM-DEMATEL), hierarchical clustering, and Preference Ranking Organization Method for Enrichment Evaluations (PROMETHEE).
In the desalination case study, the use of hierarchical clustering allows for the identification and preservation of both majority and minority opinions, based on which, the same ranking of desalination scenarios is produced through PROMETHEE. Concerning the interdependence among criteria, the results reveal that it can help identify the crucial factors for improvement of sustainability performance of the alternatives. In addition, by considering it, the understanding of criteria is enhanced, which leads to more concentrated opinions of stakeholders in the desalination case. However, the findings indicate that the influence of incorporating the criteria independency on the final decisions is restricted in cases insensitive to weight variation.
The application of BWM-DEMATEL method is a complex and time-consuming activity. Considering the effectiveness and the cost of implementation, BWM-DEMATEL might be not as worthy of application in this case study. Inspired by this, it is meaningful to develop an approach to estimate the impact of the interdependence among criteria on the alternatives ranking in a certain decision problem in the future.

Poly-hydroxy-alkanoate (PHA) is an intracellular polymer that can be used as an energy and carbon source by microorganisms. Measuring PHA is important for understanding the microbial metabolism of enhanced biological phosphorus removal (EBPR) and aerobic granular sludge (AGS) systems. There is a commonly used method to measure PHA, which is based on organic solvent extraction and gas chromatography (GC). However, there are different versions of the same method with different parameters, but the role of some of these parameters is unclear. When different types of biomass are analyzed, there is a requirement to understand the parameters and obtain an optimal protocol. In this study, the effect of various digestion times, different alcohols and organic solvents, and acid concentrations were tested to obtain the optimal protocol. The results showed that a minimum digestion time was required to get the maximum yield of PHA, and the time might differ when using different types of biomass. Methanol was shown to be better for GC separation than propanol. Using different organic solvents didn’t affect the final concentration, and an optimal acid concentration was required to determine by comparison. The GC temperature program optimization showed that lower oven temperature in GC is more beneficial for peak separation. From the analysis, it would be suggested to use methanol and chloroform for digestion and keep the digestion time for 24 hours.

Aerobic granular sludge (AGS) is an innovative biotechnology extensively applied for treating municipal wastewater, and it can potentially treat sugar industry wastewater. Glucose is a prevalent substrate in sugar industry wastewater; nevertheless, the effect of glucose on AGS systems remains unexplored. In this study, an AGS reactor using glucose as the sole carbon source was operated in anaerobic-aerobic cycles. The system maintained a solids retention time (SRT) of 10 days, resulting in good granulation and enhanced biological phosphorus removal (EBPR) performance. The glucose fed was rapidly taken up within 10 minutes, with a portion stored as intracellular polymers such as glycogen and poly-hydroxy-alkanoates (PHAs), while another portion underwent anaerobic fermentation to lactate and formate. The carbon balance was not completely closed, with 16% of the carbon speculated to be utilized for the production of an unidentified polymer. The microbial community consisted of diverse organisms, with Micropruina identified as the most abundant genera and Ca. Accumulibacter (a typical type of PAOs) as the second most abundant genera based on metagenomic analysis. A batch test was conducted by adding an excess of glucose, lactate, and formate, revealing that lactate was the probable substrate utilized by PAOs. Additionally, Micropruina was hypothesized to be involved in glucose consumption, glycogen storage, and lactate production. Micropruina and Ca. Accumulibacter collaborate in utilizing glucose, providing them with a significant competitive advantage within the system. Due to their slow growth rate, these bacteria play a crucial role in achieving favorable granulation when supplied with glucose. Promoting the growth of these organisms can be a valuable strategy in engineering applications.

Of all the greenhouse gases (GHGs), carbon dioxide (CO2) has been the target of most climate recovery efforts as it is the most abundantly emitted GHG by mass. In fact, in 2015 a legally binding international treaty was adopted by 196 parties in Paris, France to constrain the anthropogenic warming to 1.5-2.0˚C above the pre-industrial level. In order to meet this goal, a carbon budget was formulated as an estimate of the amount of carbon that can be emitted while limiting the anthropogenic warming to prescribed levels. However, the global CO2 emissions from industries are rapidly depleting this budget. Therefore, to mitigate the effects of climate change, CO2 emissions must be reduced by employing alternative commodities that can replace petrochemical resources. In this context, mixed culture fermentation presents an opportunity for redefining CO2 and waste streams as raw material for production of commodities traditionally derived from petrochemical resources. Previous studies by on this topic have indicated a potential association between elevated CO2 levels (pCO2) and butyrate formation from mixed culture fermentation. However, the cellular mechanism underlying this association are still poorly understood. Therefore, the principal objective of this research was to investigate the effects of initial substrate concentrations (g/L) and elevated pCO2 (bar) conditions on selectivity (moli/moltotal) of biomolecules produced from anaerobic conversion of glucose. For this purpose, a between-subject mixed factorial experimental design was developed to gauge the main and interaction effects of initial substrate concentrations (g/L) and elevated pCO2 (bar) conditions on selectivity of biomolecules. The principal findings of this research indicate that a strong positive relationship exists between the pCO2 and butyrate formation as the application of CO2 in reactor (EPBs) headspace resulted in higher butyrate selectivity compared to the control reactors (APBs). However, contrary to the conclusions reached by previous studies it was found that increasing the initial substrate concentration steered the product formation towards lactate and not butyrate. Whereas the highest recorded butyrate selectivity for EPBs was 30.41% for experimental condition with 5 g/L substrate concentration and 4 bar pCO2, the highest recorded butyrate selectivity for APBs was only 11.72% for 10 g/L substrate concentration and atmospheric pressure conditions. Conversely, the highest recorded lactate selectivity for EPBs was 15.13% for 20 g/L substrate and 3 bar pCO2 while the highest recorded lactate selectivity for APBs was 47.95% for 25 g/L substrate concentration and atmospheric pressure conditions. As a result of these investigations, theories concerning formation of butyrate and lactate were proffered in context of the role of CO2 in mixed culture fermentation. By confronting the existing understanding regarding product formation with new evidence this investigation seeks to advance theories concerning mixed culture fermentation.

Groundwater (GW) makes up roughly half of the global drinking water supply. Conventional iron removal in GW treatment produces approximately 10,000t/d of iron sludge. Iron sludge consists of low-density flocs with low to no commercial value and causes frequent energy intensive backwashing of the rapid sand filter. This study aimed to explore the novel concept of iron removal via iron sulfides formation. Iron sulfides are usually forming dense structures and offer a wider range of re-use applications.
To investigate this, an up-flow column reactor filled with pyrite seeding crystals was built and fed with iron and sulfide containing solutions. Flushed out formed solids were investigated with X-ray diffraction analysis and Raman spectroscopy. Seeding crystals were analyzed with scanning electron microscopy with energy dispersive X-ray spectroscopy.
This study observed rapid mackinawite formation after a few minutes. Mackinawite was likely retained by electrostatic adhesion on the pyrite seeding crystals. The molar ratio of removed iron to removed sulfide equaled up to 0.8 ± 0.2.
Mackinawite formation can present an interesting alternative to conventional iron removal, due to (i) its compact size, (ii) fast formation rates and (iii) possibly simple removal mechanism via electrostatic adhesion. Furthermore, in-situ formed mackinawite has the potential to simultaneously treat a wide range of pollutants ranging from toxic metals and metalloids such as arsenic over organic contaminants and nutrients such as nitrate and phosphate. Moreover, in-situ electrochemical dosing of sulfide by sulfate reduction might present a chemical-free solution for this approach. These potential synergies should be addressed in further investigations.

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...
@en

The first full-scale Kaumera extraction plants are in operation and increases the circularity of WWTP already. However, to reach a goal of zero waste production it is necessary to look into the waste stream of the Kaumera extraction itself. Roughly 30% of organics is extracted in the process and the remaining organics in the waste can be further recovered using anaerobic digestion (AD). In this study the continuation of the alkaline AD was used, instead of neutral digestion. Main reason for the alkaline digestion compared to the neutral digestion is the increase in CH4 content in the biogas as CO2 remains in the liquid at pH 9.6. The batch incubation uses the alkaline waste residuals stream of the Kaumera extraction plant in Epe. The digestion was done at haloalkaline conditions (pH 9.6; 0.6 M Na+). Combinations of inoculum enriched for similar substrates from a previous study and fresh soda lake sediment were used for the AD batch incubations. CH4 yields varied from 8-28% of total COD going into CH4. Compared to literature this is on lower side as these conversions are in the range of 35-50%. However, some were incubations with pre-treated substrate and already enriched incubations. Others
were neutral digestion of similar substrate and the substrate used in this study. This does show the potential still for a higher conversion of methane in the alkaline digestion of the Kaumera residuals. Based on a titration, the Alkalinity need to keep the pH at 9.6 is 3 g/L of NaOH to prevent a drop from pH 9.64 to 9.34. The overall process observed takes longer than the neutral digestion due to a delay seen in acetate conversion, therefore no bottle-neck in the process could be defined and only a kinetic problem was identified. This could be tackled by transferring the process toward continuous operation, avoiding the slow growth of syntrophic acetate oxidisers once steady state is achieved. The process was modelled using two different methods, where issues surrounding the pH description arise. For the models it is essential to extend the simple buffer capacity description in order to reliably simulate the pH dynamics of the system. As of now information around the microbial community is scarce making the modelling of the alkaline ADM1 a difficult task. To improve the alkaline ADM1 work should be done to determine kinetics of the microbial community and a better description of the substrate with
inoculum. The Dry matter (DM) of the process was 0.88%, which is quite low as in full-scale system usually at least 5% DM is used. The low DM used will lower the chance of bottle-necks in the process, thus with an increase of almost 6 times in DM the inhibition threshold of 420 mgNH4−N will be exceeded. Other implication that need to be solved is the highly saline and high pH waste stream after solids removal. Either this should be recycled back for subsequent digestions or added to the influent of an WWTP assuming it will be diluted enough to not have a major impact anymore. In the future continuous operation should be evaluated as an alternative strategy to prevent the syntrophs from delaying the process and operational parameters like the hydraulic retention time (HRT) and solid retention time (SRT) need to be studied for optimal digestion in such
a system.

The brine generated from desalination is a threat to the environment, and its disposal has been a great challenge. A new concept Zero liquid discharge (ZLD) can minimize the environmental impact of brine. However, the high costs for construction and running of a ZLD plant limits its growth in desalination market. Therefore, a new strategy integrating brine mining with desalination is proposed to improve its economic performance due to the valuable minerals in brine. A cost-effectiveness analysis (CEA) is carried out to assess its economic performance and compare this strategy with other ZLD strategies. The results present that the cost-effectiveness of the studied strategy is lower than ZLD systems which only maximize water recovery. The cost-effectiveness ratio of the studied strategy is 0.056€/kg of freshwater, higher than ZLD maximizing water recovery (0.032€/kg of freshwater). However, its profitability is higher than other ZLD schemes. This study shows that the integrated desalination and brine mining strategy has a great economic potential. Its cost-benefit of is 1.12, far lower than that of ZLD only maximizing water recovery (26.08). In addition, it can be indicated that CEA is not comprehensive enough to assess the economic performance of a multi-product desalination system. It doesn’t include the revenues from by-products, which are an important part of the studied strategy. For further research, a more integrated approach of economic analysis is needed to make a decision on the different alternatives of desalination.

With the increasing focus from policymakers to a circular economy, assessing the environmental impacts of circular products is becoming more important. In this thesis a Life Cycle Assessment of the new circular composite Re-plex is performed. The Re-plex can be used as building material. Re-plex is produced from Kaumera Nereda® Gum recovered from Nereda® wastewater sludge, and Recell® cellulose recovered from wastewater. Re-plex is still in the developmental phase, this LCA is performed to aid engineers to reduce the environmental impacts of the Re-plex composite. A comparative LCA is performed in which the current Re-plex production is compared to Fire-retardant Medium Density Fibreboard (FR-MDF) with the ILCD impact assessment family. The functional unit is 1 year of 1m2 use of interior finishing material.
The Re-plex has a better characterisation result in the impact category; Human Health (HH), respiratory effects, inorganics. The FR-MDF has better characterisation results in the impact categories; Climate change; Ecosystem Quality (EQ), acidification; EQ, freshwater ecotoxicity; EQ, freshwater eutrophication; EQ ionizing radiation; EQ, marine eutrophication; EQ, marine eutrophication; Human Health (HH), carcinogenic effects; HH, ionizing radiation; HH, non-carcinogenic effects; HH, ozone layer depletion; HH, photochemical ozone creation; Resources (RS), land use; and RS, mineral, fossils and renewables.
Scenarios are developed to improve the environmental performance of the Re-plex production. Increasing the amount of cellulose in Re-plex does not seem to improve the environmental performance. Three scenarios do improve the environmental performance; Replacing citric acid by succinic acid; improving the energy efficiency; and drying the Kaumera Gum before transport. These three improvements are combined in the new Re-plex scenario. The improved scenario has better characterisation results than FR-MDF in the ten impact categories; Climate change; EQ, acidification; EQ, freshwater ecotoxicity; EQ, freshwater eutrophication; EQ, marine eutrophication; EQ, marine eutrophication; HH, carcinogenic effects; HH, non-carcinogenic effects; HH, photochemical ozone creation; and HH, respiratory effects, inorganics. FR-MDF scores better in the five impact categories; EQ ionizing radiation; HH, ionizing radiation; HH, ozone layer depletion; RS, land use; and RS, mineral, fossils and renewables.
Engineers working on Re-plex are advised to change the use of citric acid to a better environmentally performing material. The environmental benefit of changing this material will add more value to a Re-plex product than the lower price when using citric acid. Further, the focus should be on improving the energy efficiency of Re-plex production and realising a lifetime of Re-plex of 32 years, similar to MDF. If these improvements can be realised, Re-plex has a better environmental performance than FR-MDF.

Groundwater is an excellent source for the production of drinking water due to its low concentration of microorganisms and contaminants. The main contaminants present in groundwater are iron, ammonium and manganese, which are sequentially removed in rapid sand filters by a combination of biological and chemical processes. Currently, limited knowledge of the removal mechanisms challenges the design of new drinking water treatment plants (DWTPs). As a result, different treatment schemes are used to treat groundwater with a similar composition. We hypothesize that the difference in plant configuration does not affect the stratification of removal processes. In the present work, the spatial distribution of taxonomic and functional microbiological profiles and physical-chemical removal mechanisms were investigated in two DWTPs with similar incoming water but different treatment schemes. One plant employs a dual bed filter, while the other uses two sequential filters. Concentration profiles and composition of the coating on the sand showed sequential removal of iron and manganese, while ammonium removal was ubiquitous. Activity batch tests revealed section-specific manganese removal mechanisms: adsorption in the first section and oxidation in the second section. The latter is the dominant process in full-scale filters. Manganese oxidation capacity remained constant over the height of the second section. Contrarily, ammonia removal was highly stratified in there. The highest ammonium removal rates were observed at the top section of the filter. Furthermore, ammonium removal rates were higher in the second section compared to the first one. In accordance, metagenomic analysis revealed higher abundances of nitrifying organisms in the second sections. Besides, co-existence of nitrifiers and iron-oxidizers was observed in the top layer, in contrast to the common opinion that iron removal has to be complete before nitrification can start. We (i) conclude that similar distributions in removal mechanisms and genomic profiles were observed in both DWTPs, regardless of plant configuration, (ii) provide the first holistic quantitative analysis of the biological and chemical reactions in full-scale rapid sand filters and, (iii) to our knowledge, prove for the very first time that iron hydroxides on the sand grains adsorb manganese under aerobic conditions, using both adsorption and desorption tests.