Microbial ecology of enhanced biological phosphorus removal from wastewater

The molecular microbial ecology of activated sludge communities carrying out enhanced biological phosphorus (P) removal (EBPR) was investigated. Activated sludges highly enriched in polyphosphate accumulating organisms (PAOs) were cultivated in laboratory scale sequencing batch reactors with an anaerobic-aerobic sequence, synthetic feed, and acetate as the primary carbon source. The community structure of these sludges was investigated using small subunit ribosomal RNA (SSU rRNA) based molecular tools, including PCR amplification of SSU rRNA genes and fluorescent in situ hybridization. SSU rRNA clone libraries were constructed from sludges with high and low non-soluble P (P_ns) contents. Comparative sequence analysis was used to determine that β-Proteobacteria closely related to Rhodocyclus spp. were present in high P_ns-content sludges. These organisms were enumerated using fluorescent in situ hybridization (FISH) and were found to be dominant (up to 70% of Bacteria) in the high P_ns-content sludges. The enzyme polyphosphate kinase (PPK), thought to be responsible for polyphosphate synthesis in PAOs, was also investigated. Degenerate primers were designed to amplify ppk genes from mixed microbial populations, and PCR-based clone libraries were constructed from laboratory scale and full scale activated sludges. In the PAO-enriched laboratory scale system, one type of ppk was dominant, as determined using quantitative dot-blot hybridization to extracted genomic DNA. This type shared 87% amino acid sequence identity with PPK from Rhodocyclus tenuis. The full length ppk gene was cloned from PAO-enriched activated sludge using inverse PCR. The enzyme was subsequently over-expressed in Escherichia coli, purified to near homogeneity, and characterized. It was found to be a true PPK, capable of synthesizing polyphosphate from ATP. Nearly identical ppk sequences were retrieved from full scale activated sludges carrying out EBPR. This sequence type was not observed in a conventional aerobic activated sludge. The molecular evolution of ppk was also investigated; five major clades of evolutionary descent were identified using phylogenetic analyses.