Dormancy is a topic that is researched extensively for various microorganisms, especially in the field of medicine. However, not many studies have been conducted for the dormancy of microorganisms in the wastewater treatment sector. Treatment processes like the NEREDA® technology use Phosphate Accumulating Organisms (PAO) to remove phosphate from the wastewater. If dormancy affects these bacteria, it can cause the wastewater treatment plants to function at a lower capacity. Reviving dormant PAOs can increase treatment capacity and treat the wastewater during high influent loading without effluent deterioration. Studying the optimal conditions for granule storage without inducing dormancy can be advantageous during granule transport for inoculation of reactors during their start-up phase. This study presents the first independent assessment of the possible dormancy of PAOs in Aerobic Granular Sludge (AGS).

In this research thesis, dormancy is a survival strategy opted by PAOs to persist substrate limitation. There are two different pathways to dormancy that has been explored here. Responsive switching and starvation are studied as they express fast and slow transition respectively into dormancy. Lag faced during resuscitation of dormant cells is considered proportional to the length of dormancy in responsive switching.

The experimental phase of the research can be interpreted in three parts: first through method development, second through method testing and third through the application of the developed method. The method was developed to understand dormancy in PAOs by observing differences in their maintenance and activity rates. This method was then tested on granules where dormancy was induced by lowering the temperature to 4ºC and anaerobically storing without feed. This method was then applied to newly sampled granules from a full-scale reactor to study dormancy in the PAOs. The developed method increased the activity of the former granules by 60% but did not raise the activity of the latter. Maintenance rates increased by 167% and 68%, respectively, in the granules. Although dormancy could not be sufficiently proved by the method, starvation of PAOs in Utrecht NEREDA® reactors was possible.

Pre-fermentation of influent has been proposed as a method to improve phosphorus removal capacity, granulation, and simultaneous nitrification denitrification for aerobic granular sludge plants. This thesis sought to determine the influence of increasing VFA concentrations on performance of aerobic granular sludge plants and key considerations for implementation of pre-fermentation. Two approaches were applied during this thesis: a modelling approach and an experimental approach. A mathematical model was built to show the influence of increasing VFA concentrations and to describe principles observed in practice at aerobic granular sludge plants. The experimental approach consisted of two measuring campaigns. First, experiments were performed to test the necessary operational conditions to ferment influent in a sequencing batch reactor fermenter. Second, a sampling campaign was conducted to determine the conditions which led to fermentation of organic matter in a sand trap on site in Utrecht. The model demonstrated the benefit of combining existing models to describe settling of aerobic granular sludge with size fractionations, plug flow feeding in a settled sludge bed, reactions in the bulk and in granules, and diffusion across granules and into bulk liquid. It was found that increasing acetate concentrations led to longer stability of phosphorus uptake capacity and that SRT control is necessary to maintain long term capacity. Increasing acetate concentrations also led to increases in phosphate release and simultaneous nitrification denitrification. These changes were attributable to differences observed across granules of different sizes: phosphate accumulating organisms dominated larger granules and nitrifiers were more dominant in smaller granules. Model calculations indicate that a maximum granule size exists because the most poly-phosphate storage was not performed by the largest granules. From the experimental work, fermentation was found to be occurring (at rates of 21 mg VFA-COD/L/h ± 15) in a hydraulically underloaded sand trap which led to significant VFA production and subsequent difficulties in fermenting in the controlled SBR reactor (which produced an average of 28 mg VFA-COD/l). More research is needed to determine the optimal operational conditions to ferment influent and to demonstrate the effect on aerobic granular sludge in practice.

Nowadays the use of aerobic granular sludge, also known as Nereda™ in municipal wastewater treatment, is applied worldwide. The next innovation in for this technology is the implementation of granular sludge in continuous flow through configuration. However, many challenges will have to be faced before this technology can be applied in full scale wastewater treatment plants. One of these is the formation and behaviour of granular sludge in continuous flow reactors. In this study the start-up and steady state behaviour of two lab-scale bioreactors were compared on growth speed, granule size distribution, influent distribution and density. One of the reactors was run with an up flow feeding through the sludge bed, as performed in Nereda™ plants. The second reactor was run with a pulse feeding, followed by an anaerobic mixing phase. By combining data from image analysis with reactor cycle measurements, batch tests, PHA determination and density measurements, a classification of the granule size distribution and COD distribution over the granules was made. A comparison between the reactors could be made, as both reactors were exposed to the same conditions, except for their anaerobic influent regime. Both reactors were fully granulated at the end of reactor operations. The bed fed reactor had a faster average growth speed of granules due to the higher COD load on the biggest granule fraction. This selected for a higher mass fraction of granules greater than 1mm in diameter in the bed fed reactor. Both reactors seemed to suffer from diffusion limitation, as oxygen limitation was observed in the bed
fed reactor, while the pulse fed reactor showed spatial COD limitation. Both reactors experienced some degree of P-limitation. The results from this study can be used to model granulation and granule metabolism of aerobic granular sludge exposed to different anaerobic contact regimes.

Treatment of wastewater with aerobic granular sludge (AGS) has major advantages compared to activated sludge. The only commercial system that uses AGS is the Nereda® batch process. The disadvantage of this system is the lack of existing infrastructure. AGS in continuous systems could be a solution to this disadvantage. That’s why the HARKOS pilot is built. This pilot is based on the enhanced biological phosphorus removal (EBPR) process. The aim of this study is to make sure this pilot runs according to the EBPR process (reactor characterization) and to apply biological selective pressure for granulation. This biological selective pressure consists out of four pillars. Prevention of COD overshoot into the aerobic zone, as much COD in the anaerobic zone to storage polymers, enrichment for bio-p organisms and compartmentalization of the reactor. It’s concluded that the pilot is characterized, that no COD overshoot is present, that more COD is stored as storage polymers, that the sludge is enriched for bio-p organisms and that the reactor is well enough compartmentalized. The biological selective pressure is applied. If enough physical selective pressure is applied granulation will start in the HARKOS pilot.