Phosphorus has been successfully eliminated from wastewater by biological techniques of enhanced biological phosphorus removal (EBPR) process, which relies on a specific microbiota of polyphosphate accumulating organisms (PAOs) that accumulate phosphate as polyphosphates (poly-P). Most methods for quantification of poly-P pools suffer from low accuracy and specificity. More powerful and implementable P-analysis tools are required for poly-P quantification, which will help in improved evaluation of processes in laboratory and full-scale EBPR systems. This study developed two methods to quantify poly-P pools by releasing the poly-P from the cell. During experimental optimization, it was observed that two different methods resulted in the highest phosphate release: acetate addition at a pH of 4.8 and exposure to EDTA solution with a concentration of 1% (w/v). Treatment with EDTA resulted in a higher amount of phosphate release from all sludge samples. This was characterized by P-release of 1.5–2.5 times higher than the control tests. In contrast, treatments with acetate addition at a low pH exhibited that P-release depended upon the types of the sludge samples. The highest P-release amount and rate were found in highly-enriched PAO sludge samples, but with fewer influences on the sludge collected from WWTP, which may be attributed to the lower fraction of PAOs in the sludge. Overall, the proposed approaches to quantify the poly-P concentration can be applied in simple, user-friendly, and cost-effective ways.

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 (> 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.

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 PO₄^3--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.

Many sources of wastewater contain sulfides, which can cause excessive growth of filamentous bacteria such as Thiothrix sp. resulting in bulking sludge in conventional activated sludge systems. Granular sludge systems could potentially also suffer from the growth of filamentous bacteria. Uptake of easily degradable COD by the relatively slow growing Ca. Accumulibacter phosphatis bacteria and the absence of strong diffusion gradients due to plug flow feeding through the settled granular sludge bed are assumed to be the dominant factors for successful granulation. Sulfides will remain after this anaerobic phase and cause growth of sulfide-consuming bacteria such as Thiothrix sp. Here we observed the impact of growth of Thiothrix sp bacteria in a laboratory aerobic granular sludge reactor by feeding a mixture of acetate and thiosulfate in the influent. Thiothrix sp, proliferated when 18% of the influent COD was due to thiosulfate, forming 51.4 ± 8.3% of the total granular biomass. Despite the strong presence of these filamentous bacteria a well settling sludge was maintained (SVI₁₀ equal to 13.3 mL/g). These results confirm that sludge morphology is not necessarily a reflection of the cell morphology of the bacteria, but is highly influence by reactor operation. It also reiterates the fact that compact biofilms are formed when the substrate consumption rate is lower than the substrate transport rate.

Sialic acids have been discovered in the extracellular polymeric substances (EPS) of seawater-adapted aerobic granular sludge (AGS). Sialic acids are a group of monosaccharides with a nine-carbon backbone, commonly found in mammalian cells and pathogenic bacteria, and frequently described to protect EPS molecules and cells from attack by proteases or glycosidases. In order to further understand the role of these compounds in AGS, lectin staining, genome analysis of the dominant bacterial species, and shielding tests were done. Fluorescence lectin bar-coding (FLBC) analysis showed an overlap with protein staining, indicating presence of sialoglycoproteins in the EPS matrix. Genome analysis gives a positive indication for putative production of sialic acids by the dominant bacteria Candidatus Accumulibacter. FT-IR analysis shows upon selective removal of sialic acids a decrease in carbohydrates, extension of the protein side chain, and exposure of penultimate sugars. Enzymatic removal of sialic acids results in the removal of galactose residues from the EPS upon subsequent treatment with β-galactosidase, indicating a linkage between galactose and sialic acid at the terminus of glycan chains. This work indicates the importance of sialic acids in the protection of penultimate sugar residues of glycoproteins in EPS, and provides basis for future research in the composition of EPS from biofilms and granular sludge.

For a stable operation, the aerobic granular sludge process requires mechanically strong granules in balance with the shear forces in the reactor. Despite a wide general interest in granular stability, the mechanical strength of both anaerobic and aerobic granular sludge received very little attention. In this study, a high-shear method for strength characterization has been evaluated for full-scale aerobic granular sludge (AGS). Abrasion times up to 90 min showed a stable abrasion rate coefficient (K), while prolonged periods of abrasion up to 24 h resulted in a decrease in abrasion rate. Larger granules have higher abrasion rate than smaller granules. No abrasion was observed at low shear rates, indicating a threshold shear rate for abrasion. Lab-scale AGS showed a lower abrasion rate than full-scale AGS. Incubation of full-scale granules in NaCl led to a decrease in abrasion rate at 25 g L⁻¹ NaCl, but incubation in 50 g L⁻¹ NaCl led to a further decrease for only half of the tested granular sludge samples.