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.

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.

Biofouling and scaling are ongoing challenges for reverse osmosis (RO) membranes application in wastewater reclamation. Adequate RO feed pretreatment is necessary for biofouling and scaling control. The objective of this thesis was evaluated the effectiveness of ion exchange treatment with weak acid cation (WAC) and strong base anion (SBA) resins columns, in series, after ultrafiltration (UF) treatment for the pretreatment of municipal wastewater treatment plant effluent in order to be used for RO desalination. Specifically, the performance of two SBA resins, the Amberlite SCAV4 Cl (SCAV4) and the Amberlite IRA458 Cl (IRA458) was assessed at three regeneration levels (120, 100 and 80 g NaCl/Lr). The effect of the different regeneration levels on anions removal (sulfate, phosphate and nitrate), total organic carbon (TOC) removal and the operational exchange capacity was researched. Moreover, the subsequent effect on RO biofouling and scaling potential was investigated based on the product water quality of the two SBA resins with bio-growth potential tests and software tests (WAVE design, PHREEQC 3 and Avista Ci), respectively. Phosphate and sulfate removal was above 97% for both resins at all regeneration levels. TOC removal of about 70% was achieved in all cases. It was also observed that sulfate, phosphate and TOC removal remained the same up to the point where product nitrate concentration reached its feed concentration. The removal of nitrate was influenced by the regeneration level. For both SBA resins, a lower regeneration level caused a higher nitrate baseline leakage in product water, hence to a lower removal. The macroporous SCAV4 found to have higher selectivity towards nitrate compared to the gel IRA458. Nevertheless, the anion selectivity order for both SCAV4 and IRA458 was HCO3- < NO3- < HPO42- < SO42-. A minor increase in the operational exchange capacity for both SBA resins was observed at higher regeneration levels. Overall, the influence of the different regeneration levels was found to be limited towards the product water quality and the operational exchange capacity. Also, both SBA resins resulted to similar product water quality. The operational exchange capacity of IRA458 at each regeneration level was higher than that of SCAV4, due to the former’s greater total exchange capacity. The RO feed water quality produced without ion exchange pretreatment (only UF) supported bacterial growth up to 54 ± 1.5 × 106 cells/mL. The RO feed water qualities produced with ion exchange pretreatment in the cases of SCAV4 and IRA458 supported bacterial growth up to 3.4 ± 0.3 × 106 cells/mL and to 1.25 ± 0.2 × 106 cells/mL, respectively. The resulted reduction in the bacterial growth potential was above 90% after ion exchange treatment with either one of the tested SBA resins. The growth-limiting nutrient for the RO feed water qualities produced by either SBA resin was phosphorus. The bacterial growth supported by the two RO feed water qualities produced with ion exchange pretreatment after extra phosphorus addition was 80% lower than that supported by the RO feed water quality produced without ion exchange. This difference suggests that both SBA resins removed a considerable fraction of assimilable organic carbon (AOC). It was concluded that ion exchange pretreatment with either one of the tested SBA resins resulted in nutrients removal (P and C) in the RO feed that lowers the biofouling potential compared to RO feed pretreatment with only UF. The software results suggest lower scaling potential for several scalant types in the RO feed after ion exchange treatment with either one of the tested SBA resins. Among them was calcium phosphate, which was found to have high scaling potential in RO feed without ion exchange treatment (only UF) either with or without anti-scalant dosing. However, the scaling potential of some silica and iron based minerals was high, thus anti-scalant dosing might be required.

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.

Antibiotic resistance is one of the biggest threats to global health, food security, and development today, leading to a growing number of difficult-to-treat infections and an economic burden. It can affect anyone of any age and in any country. It is mainly accelerated by the misuse and abuse of antibiotics, poor hygiene, and a lack of sanitation infrastructure. From the One Health concept, water is the main link connecting all the compartments where antibiotic resistance has primarily developed (human, animal, and natural environments). It carries microorganisms, pharmaceuticals such as antibiotics, floating genetic information in the form of mobile genetic elements (MGEs), and genes conferring antibiotic resistance. It is thought that bacteria found in anthropogenic barriers such as wastewater and drinking water treatment plants could play a role in transferring and disseminating resistant bacteria into the natural environment. However, the mechanisms by which bacteria can exchange via horizontal gene transfer (HGT) to further disseminate antibiotic resistance genes (ARGs) in such compartments are unknown. Natural transformation is one of the main HGT phenomena by which competent bacteria pull extracellular DNA into their cytoplasm. Still, it remains widely unknown which bacteria can use such a mechanism and under which circumstances. Unraveling the composition of such free-floating extracellular DNA (exDNA) fraction in complex systems such as wastewater is crucial to identify the environmental conditions promoting gene transfer. This thesis aims to understand further the role of exDNA in the transfer and development of antibiotic-resistant bacteria (ARBs) from complex systems. The status of released DNA from different model microorganisms after different sterilization procedures was evaluated in Chapter 2. The results showed that current sterilization methods are effective in microorganism inactivation. However, stable DNA is released from microbial cultures and ends up in sewage streams with genetic information from microorganisms originating from human and animal discharges. In Chapter 3, a method using chromatography to isolate and enrich exDNA without causing cell lysis from complex wastewater matrices like influent (9 μg exDNA was obtained out of 1 L), activated sludge (5.6 μg out of 1 L), and treated effluent (4.3 μg out of 1 L) was developed. Thus, this was necessary to profile its genetic composition. Surprisingly, results highlighted that exDNA is mainly comprised of MGEs (65%), posing a risk as the prevalence of MGEs in the extracellular fraction can indirectly promote antibiotic resistance development mainly via natural transformation. In the two field investigation chapters (Chapters 4 and 5), the transfer of ARGs and MGEs and their removal capacity in a full-scale Nereda® reactor removing nutrients with aerobic granular sludge and in chlorine-free drinking water treatment plants were evaluated. These two chapters summarize the journey that antibiotic-resistant bacteria follow toward water sanitation. Resistance determinants decreased their load reaching effluents from wastewater (1.1 log gene copies mL−1) and drinking water treatment plants (2.5 log gene copies mL−1), at least when inside active bacterial cells. It is less clear regarding exDNA since the treatment process involves cell decay and lysis that releases exDNA into the environment. After profiling the exDNA both in lab-scale and full-scale experiments, the effect of environmental factors such as increasing antibiotic concentrations was evaluated on exDNA transformation in an activated sludge enrichment in Chapter 6. We showed the feasibility of distantly-related microorganisms for DNA uptake when strong environmental pressures (≥50 mg L−1) were applied. Thus, it shows that natural transformation under environmental antibiotic concentrations may not be the driving force by which bacteria take up exDNA in complex systems. However, the focus should be on other compartments such as research facilities and pharmaceutical industrial discharges. Finally, strategies to remediate ARGs (intracellular and extracellular) and ARBs from wastewater effluents were evaluated in Chapter 7. We showed how byproducts from wastewater and drinking water treatment plants, such as sewage-sludge biochar and iron-oxide coated sands, were effective at removing ARBs and exDNA from effluent waters. Collectively, this thesis shows that the exDNA fraction from water matrices is an overlooked pool of genetic fragments containing MGEs and ARGs. Thus, these could be used as genetic material to transform competent bacteria and develop ARBs. However, exDNA transformation under environmental antibiotic concentrations is not the main mechanism by which bacteria evolve and adapt in mixed cultures. It is important to highlight that anthropogenic barriers are effective at remediating ARBs, which should redirect the focus from wastewater treatment plants and tackle the antibiotic resistance issue from multiple compartments simultaneously. @en