In the context of steering product formation in anaerobic digestion systems, the present thesis work elaborates on the potential role of elevated CO2 partial pressures as an environmental driver that may influence end-product selectivity from methane towards compounds from the carboxylic platform. As an emerging field of research, organic acid production via mixed culture fermentation is currently in an exploratory phase and the understanding of basic functional principles driving each of the biochemical conversions of interest is of vital importance.The present investigation forms part of a series of studies conjunctively aimed at elucidating the effect of elevated CO2 partial pressures on glucose fermentation, which consists of a complex network of several metabolic routes. Specifically, this thesis work focuses on the effects of CO2 partial pressure on the degradation of two key metabolites that are central and/or highly relevant to the glucose conversion pathways, namely pyruvate and butyrate.

Dating back to 10,000 B.C.E., fermentation has served as a vital tool primarily for food preservation. In the nineteenth century, Louis Pasteur put forth the proposition that the occurrence of fermentation is predominantly facilitated by the presence of microorganisms. Pasteur’s discovery spurred comprehensive studies on microorganisms, revealing their strategic roles in fermentation and microbial ecosystems. However, in striving for stable processes and consistent products, researchers largely focus on simple substrates and pure cultures, yet practical applications of fermentation face obstacles due to substrate complexity and culture maintenance. This research aims to provide insights into the intricate relationship between the microbial community and the complex substrate. It primarily focuses on the anaerobic acidogenic fermentation of cabbage, with the aim of identifying and understanding the key influences on microbial behavior and fermentation patterns. Cabbage was chosen as the feedstock for this investigation due to its historical and robust use as a substrate, specifically for the generation of lactic acid. This byproduct not only holds economic value but also presents intriguing scientific implications, such as serving as a promising raw material in biodegradable plastic manufacturing. The initial segment of our research endeavors to investigate the fermentation process and characterize the microbial community dynamics observed during the fermentation of chopped cabbage. Interestingly, our analysis of chopped cabbage fermentation revealed a triphasic progression, wherein the Bacilli class—predominant in the initial stages—facilitated lactic acid production, succeeded by Clostridia and Actinobacteria driving butyrate and propionate generation, respectively. Despite the principles of thermodynamics and ATP yield suggesting that the transformation of sugars into alternative end products, such as short-chain fatty acids, is more favourable, the production of lactic acid is frequently observed, notably in fermentations involving vegetables. State-of-the art flux balance analysis models suggest that these microorganisms achieve dominance when sugar concentration exceeds a certain threshold, leading to enhanced (Lactic Acid Bacteria) LAB growth rates. We hypothesize that by employing a novel water solubilization method to separate the cabbage into soluble and ”insoluble” fractions, it would enable us to manipulate the initial soluble organic load, under the assumption that the substrate composition maintains similarity. This, in turn, could potentially influence the growth rates of LAB. Remarkably, the data revealed Clostridia class dominating the “insoluble” cabbage, while Bacilli class predominated in the soluble cabbage fermentation at the early stages. The majority of LAB are documented to necessitate specific amino acids for their growth. A crucial aspect of our research was to elucidate whether LAB engaged in cabbage fermentation exhibits a similar dependency on the substrate. To elucidate this, we used the inoculum from cabbage fermentation to ferment solely cabbage sugars in the absence of the cabbage. The objective was to determine if the microbial community could produce similar fermentation patterns without the non-monosaccharide constituents of cabbage, thus investigating their role in LAB growth. Noteworthy, in the absence of cabbage, a divergent pattern with minimal lactic acid and lack of LAB was observed, highlighting their dependence on non-monosaccharide cabbage components. This research provided critical insights into the interactions between substrates and microbial communities, with potential applications in optimizing fermentation processes in the food industry and biochemical production. It may also lead to a better understanding of microbial behavior in different substrate conditions, providing a foundation for more efficient and sustainable fermentation.

Polyhydroxyalkanoates (PHA) are a family of biopolymers produced intracellular by a range of different bacteria.PHA have attracted widespread attention as an environmental friendly replacement of fossil-based polymers, because they have thermoplastic and/or elastomeric properties, and are also biobased and biodegradable.Moreover, the properties of PHA can be adjusted by tuning the monomeric composition of the polymer.Currently, more than 150 different monomers have been discovered which can form the building blocks of the PHA polymer. PHA production can be divided in three parts: biosynthesis, recovery, and application. The first part is the biotechnological production of bacteria with PHA inside their cell. First, an organic substrate can be anaerobically converted into volatile fatty acids (VFA). These VFA form the preferred substrate for PHA production by bacteria in the next steps. An approach to make PHA biosynthesis cost-effective is by using organic waste streams as substrate in combination with mixed microbial communities. This reduces the relatively large costs for raw materials and for sterilization of the equipment. Thus far, at least 19 pilot projects have been operated to produce PHA from municipal or industrial organic waste streams using this approach. In nearly all cases, the random copolymer poly(3- hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was produced, indicating that the biosynthesis of this specific polymer is reasonably well-established. Research on the production of other types of PHA from waste streams is still scarce (Chapter 2 and 3). The main obstacles that prevent the large-scale industrial implementation of waste-derived PHA are the recovery and the application step. First of all, the PHA recovery costs are responsible for a large fraction of the total production cost due to high energy and chemical demand. Another challenge of the PHA recovery step is to achieve a high and consistent quality product when waste is used as substrate. More research is required to predict the relationship between raw material input, process parameters, and final mechanical properties of the produced PHA accurately (Chapter 4). For the application of PHA, it appeared that introducing waste-derived PHA into the conventional plastic market is a lasting and complicated procedure. This is mainly caused by a lack of distribution channels, a lack of experience in bioplastic processing, and by the small scale at which PHA is currently produced compared to petrochemical plastics. Therefore, the market entry of waste-derived PHA could have a higher chance of success if the initial aim is not to produce bioplastics. Instead, the focus should be on new applications where minor fractions of impurities, and small variations in polymer characteristics are not regarded as problematic. Such a niche application can stimulate the introduction of waste-derived PHA into the market, while avoiding the obstacles and the complexity of the conventional plastic industry. Moreover, these applications can potentially exploit the unique properties of PHA (e.g., biodegradability) more effectively (Chapter 5). The aim of this thesis was to optimize and balance waste-derived PHA biosynthesis with recovery, and to target for a niche application of PHA in self-healing concrete. To this end, research was conducted on all parts of the value chain from waste to self-healing concrete: PHA biosynthesis (Chapter 2 and 3), PHA recovery (Chapter 4), and the application of PHA (Chapter 5). Chapter 2 investigates isobutyrate as sole carbon source for a microbial enrichment culture in comparison to its structural isomer butyrate. Isobutyrate is a VFA appearing in multiple waste valorization routes, such as anaerobic fermentation, chain elongation, and microbial electrosynthesis, but has never been assessed individually on its PHA production potential. The results reveal that the enrichment of isobutyrate has a very distinct character regarding microbial community development, PHA productivity, and even PHA composition. Although butyrate is a superior substrate in almost every aspect, this research shows that isobutyrate-rich waste streams have a noteworthy PHA producing potential. The main finding is that the dominant microorganism, a Comamonas sp., is linked to the production of a unique PHA family member, poly(3-hydroxyisobutyrate) (PHiB), up to 37% of the cell dry weight. This chapter is the first scientific report identifying microbial PHiB production, demonstrating that mixed microbial communities can be a powerful tool for discovery of new metabolic pathways and new types of polymers. In Chapter 3, another uncommon VFA was examined for PHA production, octanoate. Several enrichment strategies were tested to select for a community with a high medium-chain-length PHA (mcl-PHA) storage capacity when feeding octanoate. Based on the analysis of the metabolic pathways, the hypothesis was formulated that mcl-PHA production is more favorable under oxygen limited conditions than short-chain-length PHA (scl-PHA). This hypothesis was confirmed by bioreactor experiments showing that oxygen limitation during the PHA accumulation resulted in a higher fraction of mcl-PHA over scl-PHA (i.e., a PHA content of 76 wt% with a mcl-fraction of 0.79 with oxygen limitation, compared to a PHA content of 72 wt% with a mcl-fraction of 0.62 without oxygen limitation). Physicochemical analysis revealed that the extracted PHA could be separated efficiently into a hydroxybutyrate-rich fraction and a hydroxyhexanoate/hydroxyoctanoaterich fraction. The ratio between the two fractions could be adjusted by changing the environmental conditions. Almost all enrichments were dominated by Sphaerotilus sp. This chapter is the first scientific report that links this genus to mcl-PHA production, demonstrating that microbial enrichments can be a powerful tool to explore mcl-PHA biodiversity and to discover novel industrially relevant strains. In solvent extraction of PHA, the choice of solvent has a profound influence on many aspects of the process design. Chapter 4 provides a framework to perform a systematic solvent screening for PHBV extraction. First, a database was constructed of 35 solvents that were assessed according to six different selection criteria. Then, six solvents were chosen for further experimental analysis, including 1-butanol, 2-butanol, 2-ethyl hexanol (2-EH), dimethyl carbonate (DMC), methyl isobutyl ketone (MIBK), and acetone. The main findings are that the extractions with acetone and DMC obtained the highest yields (91-95%) with reasonably high purities (93-96%), where acetone had a key advantage of the possibility to use water as anti-solvent. Moreover, the results provided new insights in the mechanisms behind PHBV extraction by pointing out that at elevated temperatures the extraction efficiency is less determined by the solvent’s solubility parameters and more determined by the solvent size. Although case-specific factors play a role in the final solvent choice, we believe that this chapter provides a general strategy for the solvent selection process. In Chapter 5, a niche application for waste-derived PHA is proposed and tested, using it as bacterial substrate in self-healing concrete. Self-healing concrete is an established technology developed to overcome the inevitable problem of crack formation in concrete structures, by incorporating a so-called bacteriabased healing agent. Currently, this technology is hampered by the cost involved in the preparation of this healing agent. This chapter provides a proof-of-concept for the use of waste-derived PHA as bacterial substrate in healing agent. The results show that a PHA-based healing agent, produced from PHA unsuitable for thermoplastic applications, can induce crack healing in concrete specimens, thereby reducing the water permeability of the cracks significantly compared to specimens without a healing agent. For the first time these two emerging fields of engineering, waste-derived PHA and self-healing concrete, both driven by the need for environmental sustainability, are successfully linked. We foresee that this new application will facilitate the implementation of waste-derived PHA technology, while simultaneously supplying circular and potentially more affordable raw materials for self-healing concrete. Chapter 6 will provide a general discussion where overarching topics were selected for a thorough analysis. Finally, recommendation for further research are proposed and an outlook for the field is given. @en

Microbes are the gears that sustain all forms of life in planet Earth and are expected to become key players in the transition towards a more sustainable society. In this thesis, microbes from sewage sludge are used for the production of polyhydroxyalkanoates, biodegradable polymers that have the potential to replace many of the petroleum-based plastics that are used nowadays.@en

One of the main challenges society currently deals with is the depletion of fossil fuels. To navigate this issue, we must embrace the concept of circularity and turn waste into a resource. Waste streams are omnifarious and their conversion into new chemical building blocks is not always trivial. Luckily, we can take a look at nature’s problem solving skills to help us out. Because nature, in due time, always finds a solution and there is a (micro)organism for everything. But.. we can also give nature a hand by simplifying the problem. The diversity and complexity of waste streams can be reduced by using gasification, where the waste is combusted at a high temperature with small amounts of oxygen. This yields syngas, a mixture consisting of mainly carbon monoxide, carbon dioxide and hydrogen gas. Syngas can be converted chemically into i.e. ethanol, but the success of this process highly depends on the ratios of CO, CO2 and H2 and the absence of impurities in the gas. Microorganisms can deal with much more variability, making them a promising biocatalyst for the conversion of syngas to chemical building blocks. Yet, we have to understand the microorganisms to be able to work together with them in combatting climate change. The work in this thesis is aimed at increasing our understanding of two specific types of microorganisms that can help us to turn waste into new chemicals: syngas fermenting bacteria and chain elongating bacteria. Together, they can form a team that turns a C1 molecule (carbon monoxide) all the way into a C6 molecule (hexanoate). To make the team as effective as possible, we studied both team members in detail. The syngas fermenting bacterium we studied goes by the name Clostridium autoethanogenum, and is already being used at industrial scale by the company LanzaTech. For its chain-elongating counterpart, however, we used a mixed community of microorganisms that was specifically selected to perform chain elongation. We used this mixed community because the single, optimal partner for C. autoethanogenum has yet to be found. It has been established previously, by other researchers, that producing a lot of hexanoate is easiest when you feed chain elongating organisms a substrate with a high ethanol-to-acetate ratio. C. autoethanogenum naturally produces ethanol and acetate, but usually in a low ethanol-to-acetate ratio. In Chapter 2 we use a theoretical framework based on thermodynamics, as well as data from literature to understand what triggers C. autoethanogenum to make ethanol. We found that acetate conversion into ethanol is a stress response used to deal with a (too) high load of CO, which can be classified as overflow metabolism. We show that this behavior not only takes place when feeding CO alone, but also in the presence of both CO and H2, underlining its relevance in syngas fermentation processes. The stress response can be induced by tuning the operational parameters of the bioreactor, such as the CO supply rate or the growth rate. In Chapter 3 we quantify this effect in the laboratory ourselves. We use a steady-state culture of C. autoethanogenum in a chemostat bioreactor and repeatedly disturb it for periods of one hour with increasing amounts of CO in the inlet gas, up to a CO partial pressure of 1.2 atm. We see that ethanol production increases with increasing CO partial pressures, and at a pCO of 0.6 atm or higher external acetate is even consumed to sustain higher ethanol production rates. This proves that the product spectrum of syngas fermentation can be directed by changing the operational conditions. Furthemore, the experimental method that we used allowed for the identification of the CO uptake rate at each CO partial pressure, directly via the off-gas measurements. We observed biomass-specific CO uptake rates of up to –119 ± 1 mmol·gx−1·h−1, which is much higher than has previously been reported for this organism. The biomass-specific uptake rate is instrumental for obtaining an accurate mathematical description (or: kinetic model) of this microorganism, which in turn allows for more accurate bioprocess design. Chapter 4 focusses on the chain-elongating counterpart of our syngas fermenter. C. autoethanogenum prefers to grow at a pH of 5 –5.5, and most chain elongators that have been described in literature rather grow at neutral pH (± 7.0). This chapter revolves around this discrepancy. By using enrichment cultures in a sequencing batch bioreactor, we select for chain elongating microorganisms both at pH 7.0 and pH 5.5. In doing so, we establish that chain elongators can live at pH 5.5 and that a very comparable microbial community (on genus-level) develops at both pH. However, the behavior in the bioreactors was not the same. At lower pH, a significantly smaller fraction of the supplied ethanol was converted to hexanoate. Instead, more of the C4 molecule butyrate was produced, likely because it is less toxic to the microorganisms than hexanoate. This means that pH is an important parameter to control the product spectrum of chain elongation and that establishing an effective microbial team for C1-to-C6 conversion likely requires more than finding microbes with the same preferred pH. In Chapter 5 we delve into the biochemistry of chain elongating microbes. They are known to be very flexible in their metabolism, and they can deal with a wide range of ethanol-to-acetate ratios. Theoretically, this ratio could even be infinite (i.e. feeding only ethanol), which would lead to the production of only hexanoate and no butyrate. We call this ethanol-only chain elongation. This is interesting from a fundamental as well as a process design perspective. Therefore, we test whether it is also possible in practice by using well-monitored batch experiments in bioreactors. We use different initial conditions: only ethanol, ethanol and a small amount of acetate and ethanol and a small amount of butyrate. We observe in the bioreactors that ethanol-only chain elongation is possible, but that it proceeds very slowly. Beside that, the microorganisms prefer the presence of either acetate or butyrate so much that they eventually start producing these compounds from ethanol themselves when they are not available. This behavior has never been observed before, nor was it regarded as possible. In Chapter 6 we present a dataset of well-controlled bioreactor experiments in 9 different initial conditions, including the experiments described in the previous chapter. This dataset can be used to refine the current mathematical description of chain-elongating microbes. We describe the initial analysis of this dataset and how we assure its quality and usability for kinetic modelling using data reconciliation. With this reconciled dataset we test the accuracy of the currently available kinetic model. From this overall analysis we set out the next steps for the formulation of a more accurate kinetic model of chain elongating microbes in the future. Chapter 7 recapitulates the significant findings from this thesis, but more importantly provides a list of questions that still remain to be answered. These questions are grouped around three different themes to provide some structure: the inner world of microorganisms, the interactions between (communities of different) microorganisms and the design of efficient (new) bioprocesses for a more sustainable world. To conclude, I reflect on the societal role of a scientist. @en

Quickly, the show is about the start. Date: 3.5 thousand million years ago, location: planet Earth, event: life. Naturally, life is starting small, even microscopically tiny. Life in the form of microorganisms endures eons of time in which the world changes. They survived, failed, adapted, thrived, and they actually changed the world. They have seen humankind step into the light of day, and they will be there when we see no more. Microorganisms are the link between the inanimate, mineral planet and the living world. They facilitate the natural cycle of the elements. The CO2 we breathe out is transformed by phototropic algae to oxygen. The nitrogen in proteins that we eat finds its way to the nitrogen gas in the air, and back into the roots of plants through countless microorganisms. And central to our life: carbon, it is the food that we eat, the oil that we burn, and the plastics that will immortalize humans’ existence. We are life, we flourish, and like all living things, we are greedy. So greedy that we disrupted the circularity of nature. We are far from the first, nor the most successful, organism to change the face of the earth. Algae made the world aerobic, and the first trees covered the world in meters of indigestible wood for millions of years. And while nature seems to have found a new balance, those algae and trees now form the oil and coal that drive our manic existence. What differentiates us from those earlier life forms is that we can appreciate that we are running on borrowed time, as we can see the world changing, fast. Over the past century, it has become clear that we are shaping a linear society, predominantly driven by fossil fuels. If we, by contrast, could manage to convert our waste streams back into resources at the same rate that we produce them, that would chime in a new era. And even more profound is that we are living in a world shaped and dominated by microorganisms. We need to start cooperating with them for our health and prosperity, which requires a better understanding of the microbial world. And although we are making significant progress; time is ticking and we could use all the help there is. This thesis is on how we can explore and utilize 3.5 billion years of help. In the first chapter the vastness, complexity and wealth of the microbial world are introduced. It focusses on a fraction of that wealth, the specific topic of interest, which is the production of biopolymers by microbial communities. These biopolymers are important building blocks for a circular society, as they can serve as precursor to oil, plastics, food, and specialty materials. Of the many biopolymers in nature, the predominant one within this thesis are polyhydroxyalkanoates (PHA), which are produced by microorganisms as their equivalent to human fat, and can be used by us to produce bioplastics. In the second chapter our key contribution to the scientific field of microbial community research is made. A key aspect that is holding back research on microbial communities is the lack of experimental freedom to bring nature to the lab. In this work, we attempt to bring cultivation research into the 21st century with a more flexible biodiscovery cultivation platform. This chapter describes a part of the hardware and software that was developed to significantly assist parallel enrichment research in dynamic conditions, it elaborates on the bioreactor setups of 8 systems, the automatization, on-line data processing, and process modelling. We demonstrate a generalized respiration rate reconstruction tool for dynamic operated bioreactors. The setup and tools described here have facilitated over twenty research topics that were conducted during and alongside this Doctoral research. The third chapter demonstrates how the setup can be used to increase the research intensity of enrichment studies. We investigated the influence of temperature on the enrichment of PHA accumulating microbial communities, which yielded several noteworthy findings. Besides an explanation for the global temperature optimum of 30°C, we identified other competitive strategies in feast-famine enrichment systems, that of fast-growth and decay, and subsequent growth on cell lysis. Furthermore, we were able to align shifts in microbial function with microbial community shifts, and addressed important issues of reproducibility in microbial community enrichments. The results demonstrate that a rigorous experimental approach involving parallel cultivation allows for unambiguous identification of competitive strategies in microbial communities. And a major improvement with this approach is that we can pinpoint where our knowledge is lacking. The fourth chapter follows a systematic investigation of a specific surprising observation that was made possible by the close monitoring of the enrichment systems. During a study investigating the influence of pH on the enrichment of PHA accumulating microbial communities (analogous to the temperature study), we noticed markedly different microbial community structure and behavior between enrichments, that seemed solely based on the type of acid used for pH control. We demonstrated that the observed changes were not directly caused by the change in acid used for pH control, but resulted from the difference in corrosive strength of both acids and the related iron leaching from the bioreactor piping. Neither system was iron deficient, suggesting that the biological availability of iron is affected by the leaching process. Our results demonstrate that microbial competition and process development can be affected dramatically by secondary factors related to nutrient supply and bioavailability, and is way more complex than generally assumed in a single carbon substrate limited process. In chapter five, we investigate a novel enrichment process for PHA accumulating microbial communities. The strict uncoupling in time of nutrient supply of two growth nutrients is investigated. The setup was used to optimize the process by investigating the influence of (i) nitrogen or phosphorous uncoupling from carbon, (ii) increased carbon to nutrient ratios, and (iii) increased exchange ratios. The uncoupling strategy resulted in stable enrichments, that achieved 89 wt% (gPHA/gDW) in eight hours, every operational cycle, making this the most PHA rich production system to date. The proposed strict uncoupling strategy yields stable microbial communities with an unprecedented combination of PHA storing capacity, productivity, product yield, and general applicability for feed streams without nitrogen or phosphate. Chapter six looks forward on the future of microbial community research, it explores the collaborative efforts between Wageningen University and Delft University in the 24 million euro UNLOCK project, for which the work in this thesis laid a principal foundation.@en

Discharging untreated wastewater will contaminate the surface waters and can lead to spread of diseases and long term ecological damage. The most common method for treatment is by the activated sludge process. In this process, nutrients like nitrogen, phosphorus and COD are removed by bacteria grown in flocs. These bacterial flocs are separated from the treated water by settling. Due to the slow settling velocities of these flocs large settling tanks are needed. Settling tanks take up most of the required space for a wastewater treatment plant. Aerobic granular sludge is a compact technology designed to reduce area requirements, save energy while providing excellent effluent quality. Bacteria are grown in granules instead of flocs and have therefore a much higher settling velocity. This reduces the area requirement significantly. So much even so, that external settling tanks are completely omitted. To grow aerobic granules a few selection principles are needed. First, the influent is brought in contact with the biomass in an anaerobic environment. Here COD is converted by the bacteria into storage polymers. These storage polymers are then used for growth in the presence of oxygen hereby removing phosphorus and nitrogen from the bulk liquid. Secondly, a settling pressure is applied by which slow settling biomass is removed from the reactor, thus leading to the formation of granules. In previous research by the PhD students Janneke Beun (principle of aerobic granulation), Merle de Kreuk (basic process technology for granular sludge nutrient removal) and Mari Winkler and Joao Bassin (Microbiology and process engineering aspects of granular sludge) the basic concepts of the granular sludge technology were worked out. In this thesis the effects of several operational conditions on the conversion processes, formation and stability of aerobic granular sludge was studied. The quick implementation of the technology in practice also meant that several important subjects still needed further investigation. To ensure a well-functioning technology in domestic and industrial applications these subjects were studied in more detail (i.e. adsorption, effect of salinity, higher temperature and other substrates). Besides laboratory work also the start-up and performance of one of the first full-scale aerobic granular sludge reactors treating domestic wastewater is described. The ammonium adsorption properties of aerobic granular sludge, activated sludge and anammox granules have been investigated in Chapter 2. During operation of a pilot-scale aerobic granular sludge reactor, a positive relation between the ammonium influent concentration and the ammonium adsorbed was observed. Aerobic granular sludge exhibited much higher adsorption capacity compared to activated sludge and anammox granules. At an ammonium concentration of 30 mg N L-1, adsorption obtained with activated sludge and anammox granules was around 0.2 mg NH4+-N gVSS-1, while aerobic granular sludge from lab- and pilot scale exhibited an adsorption of 1.7 and 0.9 mg NH4+-N gVSS-1, respectively. No difference in the ammonium adsorption was observed in lab-scale reactors operated at different temperatures (20 and 30 ºC). In a lab-scale reactor fed with saline wastewater, we observed that the amount of ammonium adsorbed, decreased considerably when the salt concentration increased. The results indicate that adsorption or better: ion-exchange of ammonium should be incorporated into models for nitrification/denitrification, certainly when aerobic granular sludge is used. Salinity can adversely affect the performance of most biological processes involved in wastewater treatment (Chapter 3). The effect of salt (NaCl) on the main conversion processes in an aerobic granular sludge (AGS) process accomplishing simultaneous organic matter, nitrogen, and phosphate removal was evaluated in this chapter. Hereto an AGS sequencing batch reactor was subjected to different salt concentrations (0.2 to 20 g Cl- L-1). Granular structure was stable throughout the whole experimental period, although granule size decreased and a significant effluent turbidity was observed at the highest salinity tested. A weaker gel structure at higher salt concentrations was hypothesized to be the cause of such turbidity. Ammonium oxidation was not affected at any of the salt concentrations applied. However, nitrite oxidation was severely affected, especially at 20 g Cl- L-1, in which a complete inhibition was observed. Consequently, high nitrite accumulation occurred. Phosphate removal was also found to be inhibited at the highest salt concentration tested. Complementary experiments have shown that a cascade inhibition effect took place: first, the deterioration of nitrite oxidation resulted in high nitrite concentrations and this in turn resulted in a detrimental effect to polyphosphate-accumulating organisms (PAOs). By preventing the occurrence of the nitrification process and therefore avoiding the nitrite accumulation, the effect of salt concentrations on the bio-P removal process was shown to be negligible up to 13 g Cl- L-1. Salt concentrations equal to 20 g Cl- L-1 or higher in absence of nitrite also significantly reduced phosphate removal efficiency in the system. When aerobic granular sludge is applied for industrial wastewater treatment different soluble substrates can be present. For stable granular sludge formation on volatile fatty acids (e.g. acetate), production of storage polymers under anaerobic feeding conditions has been shown to be important. This prevents direct aerobic growth on readily available COD, which is thought to result in unstable granule formation. In Chapter 4 we investigate the impact of acetate, methanol, butanol, propanol, propionaldehyde and valeraldehyde on granular sludge formation at 35 °C. Methanogenic archaea, growing on methanol, were present in the aerobic granular sludge system. Methanol was completely converted to methane and carbon dioxide by the methanogenic archaeum Methanomethylovorans uponensis during the one-hour anaerobic feeding period, despite the relative high dissolved oxygen concentration (3.5 mg O2 L-1) during the subsequent two-hour aeration period. Propionaldehyde and valeraldehyde were fully disproportionated anaerobically into their corresponding carboxylic acids and alcohols. The organic acids produced were converted to storage polymers, while the alcohols (produced and from influent) were absorbed onto the granular sludge matrix and converted aerobically. Our observations show that easy biodegradable substrates not converted anaerobically into storage polymers could lead to unstable granular sludge formation. However, when the easy biodegradable COD is absorbed in the granules and/or when the substrate is converted by relatively slow growing bacteria in the aerobic period stable granulation can occur. The influence of sludge age on granular sludge formation and microbial population dynamics in a methanol- and acetate-fed aerobic granular sludge system operated at 35 °C is investigated in Chapter 5. During anaerobic feeding of the reactor, methanol was initially converted to methane by methylotrophic methanogens. These methanogens were able to withstand the relatively long aeration periods. Lowering the anaerobic solid retention time (SRT) from 17 to 8 days enabled selective removal of the methanogens and prevented unwanted methane formation. In absence of methanogens, methanol was converted aerobically, while granule formation remained stable. At high SRT-values (51 days) γ-Proteobacteria were responsible for acetate removal through anaerobic uptake and subsequent aerobic growth on storage polymers formed (so called metabolism of glycogen accumulating organisms). When lowering the SRT (24 days), Defluviicoccus-related organisms (cluster II) belonging to the α-Proteobacteria outcompeted acetate consuming γ-Proteobacteria at 35 ºC. DNA from the Defluviicoccus-related organisms in cluster II was not extracted by the standard DNA extraction method but with liquid nitrogen, which showed to be more effective. Remarkably, the two glycogen accumulating organisms (GAO) types of organisms grew separately in two clearly different types of granules. This work further highlights the potential of aerobic granular sludge systems to effectively influence the microbial communities through sludge age control in order to optimize the wastewater treatment processes. Recently, aerobic granular sludge technology has been scaled-up and implemented for industrial and municipal wastewater treatment under the trade name Nereda®. With full-scale references for industrial treatment application since 2006 and domestic sewage since 2009 only limited operating data have been presented in scientific literature so far. In this study performance, granulation and design considerations of an aerobic granular sludge plant on domestic wastewater at the WWTP Garmerwolde, the Netherlands were analysed (Chapter 6). After a start-up period of approximately 5 months, a robust and stable granule bed (> 8 g L-1) was formed and could be maintained thereafter, with a sludge volume index after 5 minutes settling of 45 mL g-1. The granular sludge consisted for more than 80 % of granules larger than 0.2 mm and more than 60 % larger than 1 mm. Effluent requirements (7 mg N L-1 and 1 mg P L-1) were easily met during summer and winter. Maximum volumetric conversion rates for nitrogen and phosphorus were respectively 0.17 and 0.24 kg (m3 d)-1. The energy usage was 13.9 kWh (PE150∙year)-1 that is 58 – 63 % lower than the average conventional activated sludge treatment plant in the Netherlands. Finally, this study demonstrated that aerobic granular sludge technology can effectively be implemented for the treatment of domestic wastewater. @en

With its rapidly rising concentration in the atmosphere and its high global warming potential, N2O is arguably the greenhouse gas of the 21st century. The research carried out within the Nitrous Oxide Research Alliance (NORA) – of which this thesis forms part of – focused on the microbial conversions of N2O within the nitrogen cycle, the ultimate aim being to develop N2O mitigation strategies for natural and managed ecosystems such as agricultural soils and wastewater treatment plants. A variety of pathways in the nitrogen cycle produce N2O, but respiratory N2O reduction to N2 by microorganisms harboring an N2O reductase enzyme (encoded by the gene nosZ), is the only known microbial conversion that consumes N2O. N2O-respiring microorganisms may thus be key in this endeavour. Studies in literature reporting the cultivation of denitrifying bacteria with N2O as a sole electron acceptor date back to the 1950s and in recent years, there have been important discoveries of novel groups of denitrifying and non-denitrifying N2O reducing bacteria and archaea, and their importance for N2O reduction in the environment. Nevertheless, essential aspects of N2O reduction remain unclear and the aim of this thesis was to fill in some of the existing knowledge gaps regarding N2O-reducer ecophysiology, using wastewater treatment as a frame of reference. Our main approach was to study simplified, naturally selected, N2O reducing bacterial communities in chemostat enrichment cultures fed with N2O as the sole electron acceptor and acetate as electron donor. Continuous cultivation, which selects for a fairly simple community, is ideal for ecophysiology studies as it bridges the gap between ecosystem studies and pure culture work. Furthermore, it allows for cultivation under constant and limiting conditions...@en