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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A transition to a circular economy is necessary to mitigate the negative effects on the environment of the exploitation and disposal of materials and to achieve society-wide benefits. The current produce-waste-dispose model is slowly changing toward a more sustainable produce-userecycle- upcycle model. In this context, bio-based processes using microbial mixed cultures are crucial to develop waste-to-resource valorization processes.
Purple phototrophic bacteria (PPB) form a guild of hyper-versatile organisms found in almost all aqueous environments, thriving on infrared light energy, capturing organics by photoorganoheterotrophy, and even recycling CO2 by photolithoautotrophy. Due to their outstanding metabolic versatility, their organic and nutrient capture ability, and their biomass yields over substrate approaching 1 g CODx g-1 CODs, PPB are dedicated organisms to study and use for the development of water resource recovery applications. Despite already 80 years of research on PPB, their physiology still needs to get deciphered, and their environmental biotechnological exploitation is at its infancy.
The aim of this thesis was to study and harness the metabolic versatility of PPB at different levels, from the elucidation of light-driven physiologies in pure cultures to the management of selection phenomena, population dynamics, and distributed metabolic functionalities in mixed cultures. The findings were aggregated to derive to mixed-culture bioprocess application perspectives for capturing organics and nutrients from municipal sewage and agri-food wastewater and producing valuable products, as bioplastics, biohydrogen or photopigments. In this thesis, a comprehensive overview of the potential of PPB for water resource recovery is given. The molecular principles and ecological dynamics governing the PPB metabolism were elucidated with the goal to demonstrate the potential of PPB-based biotechnologies. ... Read more

Context: The study and analysis of (meta-)genomics have been providing scientists with valuable insights into the functioning and composition of microbial communities. Latest advancements in next-gen and high throughput sequencing technologies have resulted in significant growth in the data produced and made available for further research. These advancements can help scientists dive deeper into the analysis of uncultivated microbial populations that may have important roles in their environments. Gap: However, analysis of such data requires multiple preprocessing and computational steps to interpret the microbial and genetic composition of samples. For most researchers, configuring these tools, linking them with advanced binning and annotation tools, and maintaining the provenance of the processing continues to be extremely challenging. Moreover, the most common issue with current practices of metagenomics is the reproducibility of the research due to the complexity of setup and configurations. Aim: Our aim is to get a big-picture understanding of the common practices and approaches for metagenomic analyses and to find out which ones are more often used by researchers and why. Further, to compare some of the existing tools and look into possibilities of developing and/or using a reproducible pipeline and give some general recommendations for it. Methods: For this purpose, three main methods were used. First, a literature survey was performed on metagenomic analysis approaches, methodologies, and tools. Next, researchers and scientists with different educational backgrounds active in this field were interviewed. Lastly, the process of pipeline construction and bottlenecks were evaluated through hands-on experience. Findings: By conducting this research, several common pitfalls and shortcomings of metagenomic analysis practices were identified. Since the expertise of most researchers in this field is lacking a fundamental computer science and programming background, very few would attempt developing a pipeline from scratch. Therefore, if instead, they would opt for using “ready-made” General Purpose Pipelines (GPP), they would also face various difficulties in setting up and configuring them to their needs. Also, it has been observed that many of the existing metagenomic tools are not developed and maintained according to computer science code production standards. Therefore, even the more popular tools can suffer from detrimental bugs that can render them broken and consequently deprecated. However, with the emergence of the new “all-in-one” interface-based online platforms such as Kbase.us that enable simple point-and-click set-up and sharing of workflow, there is hope for entering a new era of reproducible metagenomic analysis. ... Read more

Purple non-sulphur bacteria (PNSB) are phototrophic bacteria currently under study in the wastewater treatment sector due to their performant nutrient and resource recovery. Until recently, most of researches have focused on closed anaerobic photobioreactors resulting in high selectivity of PNSB and appealing hydrogen, microbial proteins, and carotenoids productivity. Unfortunately, these researches also showed that the implementation of this technology is hampered by its relatively high costs. As is the case for microalgae technology, raceway reactors could possibly overcome this problem thanks to their low investment and maintenance costs. However, a survey of the scientific literature shows that only few researches have investigated the application of PNSB technology with open raceway reactors and that the limited available mechanistic models do not consider the specific conditions which characterize these reactors. Therefore, this study aims to construct a mechanistic mathematical model which includes the mixotrophic metabolism of PNSB and could be used to predict the nutrient removal and recovery and PNSB relative abundance in the raceway reactor. The model was structured mainly considering the photoorganoheterotrophic and respiring chemoorganoheterotrophic growth of PNSB competing with standard (an)aerobic-respiring and fermenting chemoorganoheterophic bacteria under semi-aerobic conditions. The model was tuned with seven batch and sequencing batch reactor (SBR) laboratory experiments. It simulates the reactor performance (COD removal rate 480-780 mgCOD/L/d, yield 0.40– 0.65 mgCODx/mgCODs) and the relative PNSB abundance (10-60%) under different operational conditions (light, dissolved oxygen, surface area) with a relative error around ± 20%. This research proposes an one-at-the-time sensitivity analysis, analysing the impact of those variables (TSS, SRT, COD, light, and biotic competition) which could play an important role in real-case scenarios. From this analysis, it emerged that influent suspended solids (TSS>250 mgTSS/L), hourly variations of the natural light cycle intensity and drops of the available soluble substrate (COD<1000mgCOD/L) could strongly disturb the abundance of PNSB in the system (from 48% to 10%). An extension of the sludge retention time, from 2 to 5 days, was observed to favour the relative abundance of PNSB (from 48% to 60%) and to increase the TSS productivity (from 235 to 400 mgTSS/L/d). The model was intended as a first attempt to simulate the nutrient removal and the PNSB dynamic in a raceway reactor. It has the flexibility to study the impact of the crucial parameters evidenced from the literature review (light, oxygen, carbon source). However, several implementations will be needed in the future, mainly focusing on anoxic chemoheterotrophic growth and hydrolysis. ... Read more

The rise of antibiotic resistant bacteria threatens the existing status quo of successful treatment of infectious diseases, leading to substantial personal and economic losses. Wastewater, carrying antibiotic resistant microorganisms from fecal origin, is an important route for disseminating anthropogenic-related resistant bacteria to natural ecosystems. Wastewater treatment plants (WWTPs), collecting and treating sewage, comprise an opportunity to mitigate such dissemination. However, because of their intrinsic characteristics, namely constant nutrient inputs, presence of selectors in sewage (i.e., antibiotics), and high bacterial densities within the biological treatment, these facilities have been postulated as environments selecting for antibiotic resistant bacteria and fostering horizontal exchange of antibiotic resistance genes (ARGs). Unravelling the ecology of antibiotic resistant determinants in WWTPs is essential to identify which stages or technologies are critical for their proliferation or removal and pinpoint possible additional or alternative intervention strategies. This thesis aims to contribute to such a quest with a multidimensional approach. The work presented here involves extensive field studies combined with qPCR measurements and statistical analysis to assess how WWTPs affect antibiotic resistant determinants. In addition, culture and molecular assays are used to investigate the conjugal exchange of plasmid-borne antibiotic resistance in wastewater environments. ... Read more

Anaerobic membrane bioreactor (AnMBR) is a promising technology to treat phenolic wastewater. Conductive materials such as magnetite and granular activated carbon have been reported to be capable of improving anaerobic digestion by facilitating direct interspecies electron transfer (DIET). This research first investigated the effect of magnetite on the treatment of synthetic p-cresol (a relative abundant compound in phenolic wastewater) wastewater in a lab-scale AnMBR. Magnetite increased the reactor stability, permitted higher p-cresol loading rate in the AnMBR, and reduced the fouling potential of supernatant of the mixed liquor. Activities of dehydrogenase and F420 were significantly increased and this may have contributed to the enhanced reactor performance. Magnetite supplement did not have a substantial influence on the soluble microbial products (SMPs) concentration compared to the stage without magnetite whereas extracellular polymeric substances (EPS) concentration significantly increased with magnetite supplement. Reduced fouling potential of the supernatant of the mixed liquor may be attributed to the decrease of protein content in SMPs in the stage with magnetite supplement. Second, the effect of magnetite on the methanogenic degradation pathway of p-cresol was studied, in which the rate limiting step was the conversion of intermediate compound benzoate. Moreover, magnetite increased the maximum substrate degradation rate of all the chosen intermediates as well as the accumulative methane production. Batch test using inoculum adapted to magnetite failed to yield faster substrate degradation rate in comparison with the batch test using non-adapted inoculum. This may be ascribed to the loss of biomass when magnetite was removed from the collected sludge because magnetite and DIET-based microorganisms were closely associated and shaking manually was not sufficient for microorganisms to detach from the magnetite. Since magnetite nanoparticles enhanced reactor performance and stability as well as reduced fouling potential of the supernatant of the mixed liquor, potential commercial application of magnetite nanoparticles in AnMBR may permit shorter hydraulic retention time (HRT) and higher flux, which can lead to higher treatment capacity and lower operational costs. Further research should investigate the effect of potential magnetite corrosion on the reactor performance, the effect of magnetite on fouling potential of the mixed liquor, and likely loss of biomass in case of magnetite removal and methods to remove magnetite with as little loss of biomass as possible. ... Read more

The performance of AGS reactors treating municipal wastewater can be optimised by converting influent particulate matter into readily available substrate. This can be done via anaerobic hydrolysis and fermentation of the influent. Anaerobic processes taking place in pressure sewers are not fully understood but show the potential to act as a pre-treatment for the wastewater reaching AGS reactors. Moreover, the contribution of the influent to the hydrolytic activity of the reactor is unknown. This research evaluated the impact of a pressure sewer on wastewater characteristics, as a possible pre-treatment of sewage before reaching the treatment plant. The variations of sewage in terms of physicochemical composition and microbial activity were monitored in a full-scale pressure sewer, focusing on the hydrolysis and fermentation of organic matter for further treatment in AGS reactors. Moreover, the contribution of the influent to the enzymatic activity of a full-scale AGS reactor was assessed.
Inaccuracies deriving from sampling on a full-scale pressure sewer might have affected the results. However, statistical analyses helped to derive trends from the collected data. The pressure sewer primarily affected the degree of fermentation of the wastewater and the concentration of suspended solids. It is hypothesised that such variations could benefit the performance of AGS reactors. Although the biodegradability and enzymatic activity of the wastewater did not improve significantly, anaerobic conveyance seemed more appropriate than aerobic transport for AGS reactors. However, the influent did not seem to have a large contribution to the total reactor activity, due to the high concentration of granular biomass. ... Read more

Galdieria sulphuraria (G. sulphuraria ) is a eukaryotic, extremophilic, spherical and unicellular species of red algae. G. sulphuraria can grow at very low pH-values (pH 0.05 – 5.0) and high temperatures (35 – 56 °C). The growth conditions of G. sulphuraria make it suitable for axenic cultivation because the low pH and high temperature minimalize the risk of microbial contamination. Next to its ability to remove nutrients in wastewater treatment, G. sulphuraria is a prospective producer of a valuable product, Phycocyanin (PC), a thermostable blue pigment-protein complex, which is used as, among others food additive and food colorant.

These characteristics of G. sulphuraria lead its selection by Evides Industriewater for the uptake of the ammonium present in the reverse osmosis (RO) concentrate of New Energy and REsources from Urban Sanitation (NEREUS). NEREUS focuses on the re-use of nutrients present in wastewater, among others ammonium. One of the goals of NEREUS is to re-use the ammonium present in the RO concentrate with the use of algae. In order to recover ammonium from the RO concentrate of NEREUS, it is necessary to test whether G. sulphuraria is capable of growing on such medium. The possibility of cultivating of G. sulphuraria on the RO concentrate from water and resource recovery pilot plant of NEREUS was investigated in this thesis.

The objectives of this thesis were to find the optimal growing conditions and assess the biomass growth and nutrients consumption. Screening experiments with synthetic Allen medium, which is usually used for the cultivation of the G. sulphuraria, were conducted to obtain the best growing conditions of G. sulphuraria. In order to understand the best growing conditions for the cultivation of G. sulphuraria, the effects of several factors were investigated, which are: 1) different metabolism, 2) different nitrogen sources and concentrations (ammonium: 100 – 1000 mgNH4+-N/L and nitrate: 247 mgNO3--N/L), 3) different carbon sources (glucose, bicarbonate and CO2) and different glucose concentrations (C:N = 5:1 and 10:1), 4) different phosphate concentrations (N:P = 37:1 and 7.2:1), 5) culture densities. Ammonium with mixotrophic metabolism turned out to be the best nitrogen source. Biomass concentration on ammonium was four times higher than on nitrate. Increasing the ammonium concentration from 200 mgNH4+-N/L to 1000 mgNH4+-N/L resulted in around 25% more biomass and no firm conclusions could be drawn from the experiment performed with different phosphate concentrations. No significant increase in the growth of G. sulphuraria was observed between Carbon:Nitrogen (C:N) ratio = 5:1 and 10:1. Furthermore, culture densities higher than 0.7 g/L of biomass resulted to a slower growth of G. sulphuraria.

Experiment with synthetic RO concentrate shows that there was light limitation involved during the cultivation. Highest and fastest growth (µmax = 0.78 day-1) was observed in the mix of 40% real RO concentrate and 60% synthetic RO concentrate medium culture. Growth inhibition was observed in cultures containing RO concentrate of NEREUS. Still, G. sulphuraria did grow on RO concentrate of NEREUS. This work is contributing to the scientific and engineering community in the field of microalgae.
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As freshwater resources are predicted to become more scarce in the future, desalination will become a more prevalent treatment method. Microbial desalination, defined as the use of microbial processes to remove ions from a saline solution, may have potential as a new desalination method to produce water for domestic, agricultural or industrial purposes. This research explored the theory, performance, application and feasibility of microbial desalination. It consists of three research lines. Firstly, the performance and optimal conditions for microbial desalination were examined in batch experiments. In the second research line, the use of microbial ion transport proteins in a biomimetic membrane was proposed and the microbial light powered transport protein SyHr was genetically designed and expressed for this purpose. Finally, two models weremade which evaluate the feasibility ofmicrobial desalination at full scale; one which compares the costs of microbial desalination in sequencing batch reactors to seawater reverse osmosis and one to calculate the recovery and required size of a biomimetic membrane as described in the second
research line. Based on the results of all research lines, it was concluded that microbial desalination is be a promising new technology for desalination, which should be further developed. ... Read more

Green phototrophs such as microalgae and cyanobacteria have been proven to be able to perform photoorganoheterotrophic metabolism. The use of organic carbon can result in higher biomass production and increased concentrations of valuable compounds. Organic waste streams can serve for this purpose and lower the biomass production costs. Cheese whey, a by-product of the dairy industry with a high organic content, can be a suitable organic carbon source.
Previous studies have focused on axenic cultures, determining the species that can perform this metabolic pathway and the best substrates for their growth. However, the use of organic substrates by phototrophs in mixed microbial communities is less understood, as it is the result of multiple metabolic processes. As mixed cultures are economically preferable to pure ones, further research is needed to understand the competition mechanisms taking place in mixed-culture processes and how they can be engineered to promote the selection of phototrophs.
The first part of this work focused on the production and spectrum of volatile fatty acids from the acidogenic fermentation of 40% demineralized cheese whey. The maximum degree of acidification (77±7%) of 40% demineralized cheese whey (DWP40) was obtained when thermal (90°C) pre-treatment of the inoculum was applied and combined with a F/M ratio of 0.5 g COD/g VS.
The second part aimed to assess the selection for green phototrophs in a photoorganoheterotrophic mixed culture, using organic carbon sources derived from cheese whey, namely DWP40, lactose (as a model constituent of cheese whey), and acetate (as model volatile fatty acid derived from acidogenic fermentation of cheese whey). The cultivations were carried out in shake-flasks prior to implementation in a continuous-flow stirred-tank photobioreactor. DWP40, lactose, and acetate sustained the growth of green phototrophs in the mixed culture. Amongst the organic carbon sources, the use of acetate resulted in the highest biomass growth (170 mg VSS/L) and pigment content (87 μg/mg VSS). The selection for phototrophic organisms was possible both in batch and continuous mode.
The results obtained showed that the conversion of the lactose inside cheese whey to acetate could improve its uptake by phototrophs. Their selection inside a photoorganoheterotrophic mixed culture can be improved by higher pH and inorganic nutrient concentrations, and lower dissolved oxygen levels. ... Read more

The presence of extracellular DNA (eDNA) containing antibiotic resistance genes in the treated wastewater effluents can contribute to the spread of antimicrobial resistance (AMR) among receiving waters. The removal of cell associated antibiotic resistance genes (ARGs) has been widely studied using advance treatments. However, these treatments were not evaluated for cell free or extracellular ARGs resulting from the cell lysis or secretion during metabolic activities. eDNA is known to well adsorb onto clay, suspended particles and other soil components. Thus, in this research the potential and the main mechanisms involved in the removal of eDNA by adsorption onto sewage-based biochar and iron-oxide-coated sands has been studied. ... Read more

Purple non-sulfur bacteria (PNSB) consist of wide genera of phototrophic bacteria found in various aquatic system. A high versatility in their mode of growth ranging from photoheterotrophic to dark fermentation gives them various potential applications. Their nature of being phototrophs require them to obtain light as energy source for growth and for that they require photopigments essential for light to ATP conversion. In contrast to pure culture of PNSB, the effect of different illumination on mixed culture PNSB is partly revealed. The response of altering light intensity on mixed culture of PNSB and the change of photopigments were to be answered in this study. Mixed culture of PNSB were grown in a sequencing batch reactor (SBR) operation of 8 h cycle. Four different light intensity settings were used to identify the effect of light on their growth and nutrient removal rate. Light distribution model was constructed to simulate the growth and nutrient removal of pure culture Rhodopseudomonas palustris in order to compare with mixed culture PNSB results. To track the growth and nutrient removal trend, biomass and nutrients (acetate, ammonium, and phosphate) concentrations at different points during reaction phase were measured. Bacteriochlorphyll content as major photopigment was analyzed with two different methods; micro well plate reader and reverse phased HPLC. It was shown that both growth rate and nutrient removal rate were significantly diminished as light intensity decreased. The decrease of growth rate with decreasing light intensity indicated that with the given conditions, light intensity was the growth rate-limiting
factor for the mixed culture PNSB studied. In comparison to simulation results, it was shown that the mixed culture PNSB was showing faster growth and higher removal rate. Bacteriochlorophyll content in the micro plate reader showed an increase to 75% intensity condition and then a decrease to 25% intensity conditions. Bacteriochlrophyll content analyzed by the two different methods showed relatively high deviation between each other as large error in both analyzing methods were present. The effect of changing light intensity on mixed culture PNSB with respect to its growth, nutrient removal and pigment content was studied. Further investigation on both higher and lower light intensity to identify the photo-inhibition level and saturation level is necessary. For a better approximation of the simulation to experimental data,
further works on precise measurement of light distribution in the photobioreactor is required. Photo-pigment analysis methods should be either replaced by a spectrophotometer using standardized cuvettes to minimize measurement error or be optimized to obtain reliable data. ... Read more