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.

The development of rapid detection assays of cell viability is essential for monitoring the microbiological quality of water systems. Coupling propidium monoazide with quantitative PCR (PMA-qPCR) has been successfully applied in different studies for the detection and quantification of viable cells in small-volume samples (0.25-1.00 mL), but it has not been evaluated sufficiently in marine environments or in large-volume samples. In this study, we successfully integrated blue light-emitting diodes for photoactivating PMA and membrane filtration into the PMA-qPCR assay for the rapid detection and quantification of viable Enterococcus faecalis cells in 10-mL samples of marine waters. The assay was optimized in phosphate-buffered saline and seawater, reducing the qPCR signal of heat-killed E. faecalis cells by 4 log10 and 3 log10 units, respectively. Results suggest that high total dissolved solid concentration (32 g/L) in seawater can reduce PMA activity. Optimal PMA-qPCR standard curves with a 6-log dynamic range and detection limit of 10(2) cells/mL were generated for quantifying viable E. faecalis cells in marine waters. The developed assay was compared with the standard membrane filter (MF) method by quantifying viable E. faecalis cells in seawater samples exposed to solar radiation. The results of the developed PMA-qPCR assay did not match that of the standard MF method. This difference in the results reflects the different physiological states of E. faecalis cells in seawater. In conclusion, the developed assay is a rapid (∼5 h) method for the quantification of viable E. faecalis cells in marine recreational waters, which should be further improved and tested in different seawater settings.