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Emerging pollutants, such as pharmaceuticals and microplastics have become a pressing concern due to their widespread presence and potential impacts on ecological systems. To assess the ecosystem-level effects of these pollutants within a multi-stressor context, we simulated real-world conditions by exposing a near-natural multi-trophic aquatic food web to a gradient of environmentally relevant concentrations of fluoxetine and microplastics in large mesocosms over a period of more than three months. We measured the biomass and abundance of different trophic groups, as well as ecological functions such as nutrient availability and decomposition rate. To explore the mechanisms underlying potential community and ecosystem-level effects, we also performed behavioral assays focusing on locomotion parameters as a response variable in three species: Daphnia magna (zooplankton prey), Chaoborus flavicans larvae (invertebrate pelagic predator of zooplankton) and Asellus aquaticus (benthic macroinvertebrate), using water from the mesocosms. Our mesocosm results demonstrate that presence of microplastics governs the response in phytoplankton biomass, with a weak non-monotonic dose-response relationship due to the interaction between microplastics and fluoxetine. However, exposure to fluoxetine evoked a strong non-monotonic dose-response in zooplankton abundance and microbial decomposition rate of plant material. In the behavioral assays, the locomotion of zooplankton prey D. magna showed a similar non-monotonic response primarily induced by fluoxetine. Its predator C. flavicans, however, showed a significant non-monotonic response governed by both microplastics and fluoxetine. The behavior of the decomposer A. aquaticus significantly decreased at higher fluoxetine concentrations, potentially leading to reduced decomposition rates near the sediment. Our study demonstrates that effects observed upon short-term exposure result in more pronounced ecosystem-level effects following c

Photogranules are spherical aggregates formed of complex phototrophic ecosystems with potential for “aeration-free” wastewater treatment. Photogranules from a sequencing batch reactor were investigated by fluorescence microscopy, 16S/18S rRNA gene amplicon sequencing, microsensors, and stable- and radioisotope incubations to determine the granules’ composition, nutrient distribution, and light, carbon, and nitrogen budgets. The photogranules were biologically and chemically stratified, with filamentous cyanobacteria arranged in discrete layers and forming a scaffold to which other organisms were attached. Oxygen, nitrate, and light gradients were also detectable. Photosynthetic activity and nitrification were both predominantly restricted to the outer 500 µm, but while photosynthesis was relatively insensitive to the oxygen and nutrient (ammonium, phosphate, acetate) concentrations tested, nitrification was highly sensitive. Oxygen was cycled internally, with oxygen produced through photosynthesis rapidly consumed by aerobic respiration and nitrification. Oxygen production and consumption were well balanced. Similarly, nitrogen was cycled through paired nitrification and denitrification, and carbon was exchanged through photosynthesis and respiration. Our findings highlight that photogranules are complete, complex ecosystems with multiple linked nutrient cycles and will aid engineering decisions in photogranular wastewater treatment.

Wastewater characteristics can vary significantly, and in some municipal wastewaters the N:P ratio is as low as 5 resulting in nitrogen limiting conditions. In this study, the microbial community, function, and morphology of photogranules under nitrogen replete (N+) and limiting (N-) conditions was assessed in sequencing batch reactors. Photogranules under N- condition were nitrogen deprived 2/3 of a batch cycle duration. Surprisingly, this nitrogen limitation had no adverse effect on biomass productivity. Moreover, phosphorus and COD removal were similar to their removal under N+ conditions. Although performance was similar, the difference in granule morphology was obvious. While N+ photogranules were dense and structurally confined, N- photogranules showed loose structures with occasional voids. Microbial community analysis revealed high abundance of cyanobacteria capable of N2-fixation. These were higher at N- (38%) than N+ (29%) treatments, showing that photogranules could adjust and maintain treatment performance and high biomass productivity by means of N2-fixation.

Photogranules are a novel wastewater treatment technology that can utilize the sun's energy to treat (waste)water and at the same time generate valuable biomass. The microbial community consists of phototrophs (e.g.., microalgae, cyanobacteria) and non-phototrophic microorganisms (e.g., nitrifiers, denitrifiers, PAOs) that form dark-green spheroid agglomerates. Photogranules have excellent settling properties and are easy to harvest. Introducing photosynthesis to the treatment process has the advantage of in-situ oxygenation. Oxygen produced by phototrophs is directly available for heterotrophs (and nitrifiers). In turn, carbon dioxide produced by respiration is sequestered via photosynthesis. This reduces (or eliminates) the need for energy intensive external aeration and simultaneously minimizes greenhouse gas emissions. Additionally, photogranules can use both light and organic carbon for metabolic energy and consequently can have higher biomass productivity compared to conventional treatment systems. This makes them especially suitable for nutrient recovery application.The aim or this thesis was to advance the field of phototrophic wastewater treatment by systematically exploring the microbial community assembly and functions of photogranules. To achieve this, we operated bioreactors that allowed photogranulation and subsequently explored the functional potential of photogranules for wastewater treatment by changing operation conditions. We integrated various approaches and techniques to investigate the microbial community composition and function, physical structure, and microscale functionality of photogranules. Ultimately, we provided a holistic overview on the microbial ecology and process engineering of photogranular technology to pave the way for more sustainable wastewater treatment.In chapter 2 we generated photogranules under hydrodynamic conditions in lab-scale bioreactors operated in sequencing batch mode. We used a diverse and species-rich inoculum f

Background: Wastewater is considered as a renewable resource water and energy. An advantage of decentralized sanitation systems is the separation of the blackwater (BW) stream, which is highly contaminated with human pathogens, from the remaining household water. However, the composition and functions of the microbial community in BW are not known. In this study, we used shotgun metagenomics to assess the dynamics of microbial community structure and function throughout a new BW anaerobic digestion system installed at The Netherlands Institute of Ecology. Samples from the influent (BW), primary effluent (anaerobic digested BW), sludge and final effluent of the pilot upflow anaerobic sludge blanket (UASB) reactor and microalgae pilot tubular photobioreactor (PBR) were analyzed. Results: Our results showed a decrease in microbial richness and diversity followed by a decrease in functional complexity and co-occurrence along the different modules of the bioreactor. The microbial diversity and function decrease were reflected both changes in substrate composition and wash conditions. The most prevalent core functions in influent (BW) were related to metabolism of carbohydrates, response to chemicals and drugs, and nitrogen. The core functions in anaerobic digested BW and upflow anaerobic sludge blanket reactor were related to response to stress, viral processes and iron-sulfur metabolism. Methanogenesis-related functions were most abundant in upflow anaerobic sludge blanket reactor. Effluent from tubular photobioreactor presented high abundances of functions related to nitrogen utilization, metal ion binding and antibiotic biosynthetic processes. Interestingly, the abundance of sequences related to ‘pathogenesis’ decreased from influent BW to SP1 to effluent from tubular photobioreactor. Our wastewater treatment system also decreased potential microbial functions related to pathogenesis. Conclusions: The new sanitation system studied here fosters microb

Human activities are pushing earth beyond its natural limits, so recycling nutrients is mandatory. Microalgae are highly effective in nutrient recovery and have strong potential as a sustainable wastewater treatment technology. Here, nutrients from black water (toilet wastewater) were recovered as microalgal biomass, which was dried and assessed as a fertilizer in pot experiments compared with inorganic fertilizer. We deciphered the effects of microalgal biomass as a biofertilizer on plant growth and quality and the biological processes linked to greenhouse gas (GHG) emissions. In addition, we elucidated the assembly of the active microbiome in bulk soil and rhizosphere during barley development. Microalgal biomass application and inorganic fertilizer (NPK) resulted in similar plant productivity (16.6 g pot−1). Cumulative nitrous oxide (N2O) emissions were 4.6-fold higher in the treatment amended with microalgal fertilizer (3.1% of applied N) than that with inorganic fertilizer (0.5% of applied N). Nitrification by bacteria was likely the main pathway responsible for N2O emissions (R2 = 0.7, p ≤ 0.001). The application of nitrogen fertilizers affected the structures of both the active bacterial and protozoan communities, but these effects were less obvious than the strong plant effect, as the recruited microbiota varied among different plant developmental stages. Both treatments enriched similar bacterial and protozoan taxonomic orders but with different distributions through time across the plant developmental stages. Furthermore, the bacterial community showed a clear trend of resilience from the beginning of the experiment until harvest, which was not observed for protozoa. Our results indicate that the use of microalgal biomass as a fertilizer is a viable option for recycling nutrients from wastewater into plant production.

This study demonstrates that microalgae can effectively recover all P and N from anaerobically treated black water (toilet wastewater). Thus, enabling the removal of nutrients from the black water and the generation of a valuable algae product in one step. Screening experiments with green microalgae and cyanobacteria showed that all tested green microalgae species successfully grew on anaerobically treated black water. In a subsequent controlled experiment in flat-panel photobioreactors, Chlorella sorokiniana was able to remove 100% of the phosphorus and nitrogen from the medium. Phosphorus was depleted within 4 days while nitrogen took 12 days to reach depletion. The phosphorus and nitrogen removal rates during the initial linear growth phase were 17 and 122 mg·L−1·d−1, respectively. After this initial phase, the phosphorus was depleted. The nitrogen removal rate continued to decrease in the second phase, resulting in an overall removal rate of 80 mg·L−1·d−1. The biomass concentration at the end of the experiment was 11.5 g·L−1, with a P content of approximately 1% and a N content of 7.6%. This high algal biomass concentration, together with a relatively short P recovery time, is a promising finding for future post-treatment of black water while gaining valuable algal biomass for further application.