In aerobic granular sludge (AGS) systems, different-sized microbial aggregates having different solids retention time (SRT) coexist in the same reactor compartment and are subjected to the same influent wastewater. Thus, the AGS system provides a unique ecosystem to study the importance of local (species sorting) and regional (immigration) processes in bacterial community assembly. The microbial communities of different-sized aggregates (flocs <0.2 mm, small granules (0.2-1.0 mm) and large granules >1.0 mm), influent wastewater, excess sludge and effluent of a full-scale AGS plant were characterized over a steady-state operation period of 6 months. Amplicon sequencing was integrated with mass balance to determine the SRT and net growth rate of operational taxonomic units (OTUs). We found strong evidence of species sorting as opposed to immigration, which was significantly higher at short SRT (i.e., flocs and small granules) than that at long SRT (large granules). Rare OTUs in wastewater belonging to putative functional groups responsible for nitrogen and phosphorus removal were progressively enriched with an increase in microbial aggregates size. In contrast, fecal- and sewage infrastructure-derived microbes progressively decreased in relative abundance with increase in microbial aggregate size. These findings highlight the importance of AGS as a unique model ecosystem to study fundamental microbial ecology concepts.

Saline wastewater is known to affect the performance of phosphate-accumulating organisms (PAOs) in enhanced biological phosphorus removal (EBPR) process. However, studies comparing the effect of salinity on different PAO clades are lacking. In this study, 'Candidatus Accumulibacter phosphatis' Clade I and II (hereafter referred to as PAOI and PAOII) were highly enriched (~90% in relative abundance as determined by quantitative FISH) in the form of granules in two sequencing batch reactors. Anaerobic and aerobic batch experiments were conducted to evaluate the effect of salinity on the kinetics and stoichiometry of PAOI and PAOII. PAOI and PAOII communities showed different priority in using polyphosphate (poly-P) and glycogen to generate ATP in the anaerobic phase when exposed to salt, with PAOI depending more on intracellular poly-P degradation (e.g., the proportion of calculated ATP derived from poly-P increased by 5-6% at 0.256 mol/L NaCl or KCl) while PAOII on glycolysis of intracellularly stored glycogen (e.g., the proportion of calculated ATP derived from glycogen increased by 29-30% at 0.256 mol/L NaCl or KCl). In the aerobic phase, the loss of phosphate uptake capability was more pronounced in PAOII due to the higher energy cost to synthesize their larger glycogen pool compared to PAOI. For both PAOI and PAOII, aerobic conversion rates were more sensitive to salt than anaerobic conversion rates. Potassium (K⁺) and sodium (Na⁺) ions exhibited different effect regardless of the enriched PAO culture, suggesting that the composition of salt is an important factor to consider when studying the effect of salt on EBPR performance.

Salinity can affect the performance of biological wastewater treatment in terms of nutrient removal. The effect of salt on aerobic granular sludge (AGS) process in terms of granulation and nutrient removal was examined in this study. Experiments were conducted to evaluate the effect of salt (15 g/L NaCl) on granule formation and nutrient removal in AGS system started with flocculent sludge and operated at DO of 2.5 mg/L (phase I). In addition, experiments were conducted to evaluate the effect of gradually increasing the salt concentration (2.5 g/L to 15 g/L NaCl) or increasing the DO level (2.5 mg/L to 8 mg/L) on nutrient removal in AGS system started with granular sludge (phase II) taken from an AGS reactor performing well in terms of N and P removal. Although the addition of salt in phase I did not affect the granulation process, it significantly affected nutrient removal due to inhibition of ammonia oxidizing bacteria (AOB) and phosphate accumulating organisms (PAOs). Increasing the DO to 8 mg/L or adapting granules by gradually increasing the salt concentration minimized the adverse effect of salt on nitrification (phase II). However, these strategies were not successful for mitigating the effect of salt on biological phosphorus removal. No nitrite accumulation occurred in all the reactors suggesting that inhibition of biological phosphorus removal was not due to the accumulation of nitrite as previously reported. Also, glycogen accumulating organisms were shown to be more tolerant to salt than PAO II, which was the dominant PAO clade detected in this study. Future studies comparing the salinity tolerance of different PAO clades are needed to further elucidate the effect of salt on PAOs.