| Experimental methodologies to study the natural abundance of polyphosphate-accumulating microorganisms (PAO) in the enhanced biological phosphorus removal (EBPR) process were developed and optimized in this study, The first method allows the simultaneous visualization of polyphosphate granules and hybridized oligonucleotide probes, thus connecting phenotypic expression with phylogenetic identification of the microorganisms involved in phosphorus removal from wastewater. The second experimental methodology developed consisted on the separation of activated sludge microorganisms by a density gradient centrifugation. This method allows for a rapid and easy 2 to 5 fold concentration of PAO, and it permits to direct further analyses of the PAO community (i.e. cloning or culturing) on the organisms of interest. A third methodology developed to facilitate the study of PAO community in activated sludge was based on the separation of PAO and non PAO by flow cytometry. Finally, a further refinement of the separation technique was also developed using sorting of DAPI-stained cells. The sorted samples contained up to 95% PAO, making them ideal for further analyses of the PAO community.; Taken together, the methodologies developed in this study provide the experimental means to eliminate the three most common problems that have limited our ability to fully characterize the microbial ecology of the EBPR process, namely, the biases introduced by culturing methods, the inevitable change in microbial communities that accompany bench-scale experiments, and the frequent inability to connect phylogenetic identification and detection of phenotypic characteristics. |
