Physical enrichment of uncultured Accumulibacter and Nitrospira from activated sludge by unlabeled cell sorting technique

It is important to understand the ecology and physiology of microbes in activated sludge of wastewater treatment plants. Recently, molecular based approaches such as 16S rRNA genes and environmental genomics have illuminated black boxes in nutrient removal process and expanded our knowledge. However, most microbes responsible for the removal of phosphate and nitrogen such as Accumulibacter and Nitrospira remain uncultured. This is because optimum methodologies to concentrate these uncultured microbes and to obtain pure cultures have not been established. Here, we report a novel approach for physical enrichment of uncultured Accumulibacter and Nitrospira from microbial communities in activated sludge by a cell sorting system. Two scattering signatures representing forward scatter and side scatter of this system allowed morphological characterization of microbial particles in activated sludge. The distribution and size of microbial particles consisting of single cells, microcolonies, and aggregates depended on the levels of scattering signatures. Next generation sequencer and principal component analysis revealed each microbial population fractionated according to the levels of scattering signatures, resulting that uncultured Accumulibacter and Nitrospira could be sorted as single cells or microcolonies. Finally, quantitative fluorescence in situ hybridization analysis determined optimum fractions to collect sufficiently these target microbes from activated sludge. Consequently, this method would be very useful as an enrichment technique prior to isolation, genomic analysis, and physiological investigation of uncultured bacteria.