Unravelling the removal mechanisms of bacterial and viral surrogates in aerobic granular sludge systems

The aerobic granular sludge (AGS) process is an effective wastewater treatment technology for organic matter and nutrient removal that has been introduced in the market rapidly. Until now, limited information is available on AGS regarding the removal of bacterial and viral pathogenic organisms present in sewage. This study focussed on determining the relation between reactor operational conditions (plug flow feeding, turbulent aeration and settling) and physical and biological mechanisms on removing two faecal surrogates, Escherichia coli and MS2 bacteriophages. Two AGS laboratory-scale systems were separately fed with influent spiked with 1.0 × 106 CFU/100 mL of E. coli and 1.3 × 108 PFU/100 mL of MS2 bacteriophages and followed during the different operational phases. The reactors contained only granular sludge and no flocculent sludge. Both systems showed reductions in the liquid phase of 0.3 Log10 during anaerobic feeding caused by a dilution factor and attachment of the organisms on the granules. Higher removal efficiencies were achieved during aeration, approximately 1 Log10 for E. coli and 0.6 Log10 for the MS2 bacteriophages caused mainly by predation. The 18S sequencing analysis revealed high operational taxonomic units (OTUs) of free-living protozoa genera Rhogostoma and Telotrochidium concerning the whole eukaryotic community. Attached ciliates propagated after the addition of the E. coli, an active contribution of the genera Epistylis, Vorticella, and Pseudovorticella was found when the reactor reached stability. In contrast, no significant growth of predators occurred when spiking the system with MS2 bacteriophages, indicating a low contribution of protozoa on the phage removal. Settling did not contribute to the removal of the studied bacterial and viral surrogates.M.L. Barrios-Hernández acknowledges the Technological Institute of Costa Rica for providing the fellowship (Grant Number 007-2014-M) to pursue her PhD programme (2016-2020) at IHE-Delft, the Netherlands. K. Mora-Cabrera acknowledges the Generalitat Valenciana (GRISOLIAP/2017/173) and the European Social Funds (BEFPI/2019/065) for their financial support

Impact of the anaerobic feeding mode on substrate distribution in aerobic granular sludge

There is a growing interest to implement aerobic granular sludge (AGS) in existing conventional activated sludge (CAS) systems with a continuous flow-through configuration. The mode of anaerobic contact of raw sewage with the sludge is an important aspect in the adaptation of CAS systems to accommodate AGS. It remains unclear how the distribution of substrate over the sludge by a conventional anaerobic selector compares to the distribution via bottom-feeding applied in sequencing batch reactors (SBRs). This study investigated the effect of the anaerobic contact mode on the substrate (and storage) distribution by operating two lab-scale SBRs; one with the traditional bottom-feeding through a settled sludge bed similar to full-scale AGS systems, and one where the synthetic wastewater was fed as a pulse at the start of the anaerobic phase while the reactor was mixed through sparging of nitrogen gas (mimicking a plug-flow anaerobic selector in continuous flow-through systems). The distribution of the substrate over the sludge particle population was quantified via PHA analysis, combined with the obtained granule size distribution. Bottom-feeding was found to primarily direct substrate towards the large granular size classes (i.e. large volume and close to the bottom), while completely mixed pulse-feeding gives a more equal distribution of substrate over all granule sizes (i.e. surface area dependant). The anaerobic contact mode directly controls the substrate distribution over the different granule sizes, irrespective of the solids retention time of a granule as an entity. Preferential feeding of the larger granules will enhance and stabilise the granulation compared to pulse-feeding, certainly under less advantageous conditions imposed by real sewage.</p

A settling model for full-scale aerobic granular sludge

The settling behavior of aerobic granular sludge (AGS) in full-scale reactors is different from the settling of normal activated sludge. Current activated sludge models lack the features to describe the segregation of granules based on size during the settling process. This segregation plays an important role in the granulation process and therefore a better understanding of the settling is essential. The goal of this study was to model and evaluate the segregation of different granule sizes during settling and feeding in full-scale aerobic granular sludge reactors. Hereto the Patwardhan and Tien model was used. This model is an implementation of the Richardson and Zaki model, allowing for multiple classes of particles. To create the granular settling model, the most relevant parameters were identified using aerobic granular sludge from different full-scale Nereda® reactors. The settling properties of individual granules were measured as was the bulk behavior of granular sludge beds with uniform granular sludge particles. The obtained parameters were combined in a model containing multiple granule classes, which then was validated for granular sludge settling in a full-scale Nereda® reactor. In practice a hydraulic selection pressure is used to select for granular sludge. Under the same hydraulic selection pressure the model predicted that different stable granular size distributions can occur. This indicates that granular size distribution control would need a different mechanism then the hydraulic selection pressure alone. This model can be used to better understand and optimize operational parameters of AGS reactors that depend on granular sludge size, like biological nutrient removal. Furthermore insights from this model can also be used in the development of continuously fed AGS systems.BT/Environmental Biotechnolog

Nitrous oxide emission from full-scale municipal aerobic granular sludge

The nitrous oxides emission was measured over 7 months in the full-scale aerobic granular sludge plant in Dinxperlo, the Netherlands. Nitrous oxide concentrations were measured in the bulk liquid and the off-gas of the Nereda® reactor. Combined with the batch wise operation of the reactor, this gave a high information density and a better insight into N2O emission in general. The average emission factor was 0.33% based on the total nitrogen concentration in the influent. The yearly average emission factor was estimated to be between 0.25% and 0.30%. The average emission factor is comparable to continuous activated sludge plants, using flocculent sludge, and it is low compared to other sequencing batch systems. The variability in the emission factor increased when the reactor temperature was below 14 °C, showing higher emission factors during the winter period. A change in the process control in the winter period reduced the variability, reducing the emission factors to a level comparable to the summer period. Different process control might be necessary at high and low temperatures to obtain a consistently low nitrous oxide emission. Rainy weather conditions lowered the emission factor, also in the dry weather flow batches following the rainy weather batches. This was attributed to the first flush from the sewer at the start of rainy weather conditions, resulting in a temporarily increased sludge loading.BT/Environmental Biotechnolog

Aerobic granular sludge

BT/Environmental Biotechnolog

Trehalose as an osmolyte in Candidatus Accumulibacter phosphatis

Abstract: Candidatus Accumulibacter phosphatis is an important microorganism for enhanced biological phosphorus removal (EBPR). In a previous study, we found a remarkable flexibility regarding salinity, since this same microorganism could thrive in both freshwater- and seawater-based environments, but the mechanism for the tolerance to saline conditions remained unknown. Here, we identified and described the role of trehalose as an osmolyte in Ca. Accumulibacter phosphatis. A freshwater-adapted culture was exposed to a single batch cycle of hyperosmotic and hypo-osmotic shock, which led to the release of trehalose up to 5.34 mg trehalose/g volatile suspended solids (VSS). Long-term adaptation to 30% seawater-based medium in a sequencing batch reactor (SBR) gave a stable operation with complete anaerobic uptake of acetate and propionate along with phosphate release of 0.73 Pmol/Cmol, and complete aerobic uptake of phosphate. Microbial analysis showed Ca. Accumulibacter phosphatis clade I as the dominant organism in both the freshwater- and seawater-adapted cultures (&gt; 90% presence). Exposure of the seawater-adapted culture to a single batch cycle of hyperosmotic incubation and hypo-osmotic shock led to an increase in trehalose release upon hypo-osmotic shock when higher salinity is used for the hyperosmotic incubation. Maximum trehalose release upon hypo-osmotic shock was achieved after hyperosmotic incubation with 3× salinity increase relative to the salinity in the SBR adaptation reactor, resulting in the release of 11.9 mg trehalose/g VSS. Genome analysis shows the possibility of Ca. Accumulibacter phosphatis to convert glycogen into trehalose by the presence of treX, treY, and treZ genes. Addition of trehalose to the reactor led to its consumption, both during anaerobic and aerobic phases. These results indicate the flexibility of the metabolism of Ca. Accumulibacter phosphatis towards variations in salinity. Key points: • Trehalose is identified as an osmolyte in Candidatus Accumulibacter phosphatis. • Ca. Accumulibacter phosphatis can convert glycogen into trehalose. • Ca. Accumulibacter phosphatis clade I is present and active in both seawater and freshwater.BT/Environmental Biotechnolog

Biological phosphorus removal in seawater-adapted aerobic granular sludge

Seawater can be introduced or intrude in sewer systems and can thereby negatively influence biological wastewater treatment processes. Here we studied the impact of artificial seawater on the enhanced biological phosphate removal (EBPR) process performance by aerobic granular sludge (AGS) with synthetic wastewater. Process performance, granule stability and characteristics as well as microbial community of a seawater-adapted AGS system were observed. In seawater conditions strong and stable granules formed with an SVI5 of 20 mL/g and a lower abrasion coefficient than freshwater-adapted granules. Complete anaerobic uptake of acetate, anaerobic phosphate release of 59.5 ± 4.0 mg/L PO43--P (0.35 mg P/mg HAc), and an aerobic P-uptake rate of 3.1 ± 0.2 mg P/g VSS/h were achieved. The dominant phosphate accumulating organisms (PAO) were the same as for freshwater-based aerobic granular sludge systems with a very high enrichment of Ca. Accumulibacter phosphatis clade I, and complete absence of glycogen accumulating organisms. The effect of osmotic downshocks was tested by replacing influent seawater-based medium by demineralized water-based medium. A temporary decrease of the salinity in the reactor led to a decreased phosphate removal activity, while it also induced a rapid release of COD by the sludge, up to 45.5 ± 1.7 mg COD/g VSS. This is most likely attributed to the release of osmolytes by the cells. Recovery of activity was immediately after restoring the seawater feeding. This work shows that functioning of aerobic granular sludge in seawater conditions is as stable as in freshwater conditions, while past research has shown a negative effect on operation of AGS processes with NaCl-based wastewater at the same salinity as seawater.BT/Environmental Biotechnolog

Nanocellulose recovery from domestic wastewater

Wastewater solids could be an attractive source of secondary raw cellulose, mainly originating from toilet paper. Cellulose can be recovered through sieving of raw wastewater, return sludge, or excess sludge. In particular, a large fraction of cellulose (13–15%) can be found in the excess sludge of the aerobic granular sludge produced by the Nereda® wastewater technology. A cellulose extraction method was developed during this study, allowing the recovery of a pulp with over 86 wt% purity. The wastewater derived cellulose fibres could be an excellent source for production of recovered cellulose nanocrystals (rCNC). Several pre-treatment steps needed in cellulose nanocrystals (CNC) production from wood pulp are already performed in the production of toilet paper. Here, the technical feasibility of such rCNC is studied. As reference materials, microcrystalline cellulose and toilet paper were also used. The rCNC were obtained by acid hydrolysis, with yields of ∼30 wt% (pulp basis). The wastewater-based material was rod-like, with high aspect ratio (10–14), crystallinity (62–68%), and chemical structure similar to commercial CNC. The yield of rCNC per gram of cellulose recovered from the influent was 22%, while for excess sludge cellulose it was less (4%). Bio-nanocomposites of rCNC and alginate were also investigated. At 50 vol% loading of rCNC, there was a 50% relative increase in stiffness (18 GPa) compared to matrix (12 GPa). The characterization of rCNC and positive impact in composite materials confirms a suitable quality of wastewater derived CNC. Ultimately, the nanocellulose is a tangible example that recovery of high-end products from wastewater is possible, in line with a circular economy.</p