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

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

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

Increase in sea level will lead to an increase in salinity in domestic wastewater systems. In order to anticipate its effects on biological wastewater treatment, the impact has to be assessed with lab-scale experiments. Aerobic granular sludge (AGS) is a successful technology for simultaneous removal of organic carbon (COD), nitrogen, and phosphorus in a single process step. The impact
of seawater on AGS has not yet been reported. The effect of salinity on AGS can roughly be divided in three aspects: biological activity, physical stability, and change in extracellular polymeric substances (EPS). Based on these aspects, the research from this PhD thesis has been structured. The overall question is: “How does long-term exposure of seawater affect the AGS process?”. This PhD thesis shows the feasibility of the aerobic granular sludge process with seawater-based wastewater.

Cellulose is a biopolymer commonly used as a renewable constituent in the production of green materials. Wastewater solids can be an attractive source of cellulose, mainly originating from toilet paper. Cellulosic solids can be recovered (i) through sieving before the sewage treatment - resulting in a Fine Sieve Fraction (FSF), or (ii) after biological treatment in the waste sludge – such as in Nereda® Excess Sludge (NES). Its recovery and valorisation into high-end bio-based products, such as crystalline nanocellulose, could improve the techno-economic feasibility of cellulose recovery from wastewater. A cellulose extraction method from wastewater solids was developed during this project. Reference materials (microcrystalline cellulose and toilet paper) and wastewater-extracted cellulosic pulps were studied for the production of cellulose nanocrystals (CNCs). CNCs were prepared by controlled acid hydrolysis followed by centrifugation, filtration, dialysis, neutralisation and ultrasonication. A detailed protocol for CNCs isolation from toilet paper and wastewater-recovered pulps can be found in the appendix section. CNCs could be extracted at around 30 % yield (g g pulp-1). FSF showed to be an attractive source of nanocellulose since the yield per g FSF was 22 %, while for NES it was only 4 %. The wastewater-CNCs presented rod-like morphology with high aspect ratio (10 – 14), crystallinity (62 – 68 %), and chemical structure very similar to commercially available CNCs. However, there is still great extent for improvements since impurities might be still present and the isolation process needs reproducibility and optimization. Bionanocomposites consisting of CNCs (commercial and lab-made) and alginate materials were investigated by measuring the stiffness of freestanding films. A 50 % loading of commercial-CNCs reinforced alginate films by a factor of 1.6 (19 GPa). For the same loading, CNCs isolated from toilet paper and FSF reinforced alginate by a factor of 1.8 and 1.5 (22 – 18 GPa) respectively. Both characterisation results and reinforcement efficiency of CNCs isolated from toilet paper and wastewater-recovered pulps confirm the good quality of this potential product. Furthermore, we have observed changes in the nematic-isotropic phase diagram of CNCs in alginate suspensions. We developed a model hypothesizing that an alginate-shell decorates the CNCs rods increasing its volume fraction and allowing the nematic phase to appear at lower concentrations. The model was highly representative to the experimental data. Lastly, the market relevance of wastewater-CNCs is discussed together with a research outlook.