Characterization of the Denitrification-Associated Phosphorus Uptake Properties of “Candidatus Accumulibacter phosphatis” Clades in Sludge Subjected to Enhanced Biological Phosphorus Removal

Abstract

bTo characterize the denitrifying phosphorus (P) uptake properties of "Candidatus Accumulibacter phosphatis," a sequencing batch reactor (SBR) was operated with acetate. The SBR operation was gradually acclimated from anaerobic-oxic (AO) to anaerobic-anoxic-oxic (A2O) conditions by stepwise increases of nitrate concentration and the anoxic time. The communities of "Ca. Accumulibacter" and associated bacteria at the initial (AO) and final (A2O) stages were compared using 16S rRNA and polyphosphate kinase genes and using fluorescence in situ hybridization (FISH). The acclimation process led to a clear shift in the relative abundances of recognized "Ca. Accumulibacter" subpopulations from clades IIA > IA > IIF to clades IIC > IA > IIF, as well as to increases in the abundance of other associated bacteria (Dechloromonas [from 1.2% to 19.2%] and "Candidatus Competibacter phosphatis" [from 16.4% to 20.0%]), while the overall "Ca. Accumulibacter" abundance decreased (from 55.1% to 29.2%). A series of batch experiments combined with FISH/microautoradiography (MAR) analyses was performed to characterize the denitrifying P uptake properties of the "Ca. Accumulibacter" clades. In FISH/MAR experiments using slightly diluted sludge (ϳ0.5 g/liter), all "Ca. Accumulibacter" clades successfully took up phosphorus in the presence of nitrate. However, the "Ca. Accumulibacter" clades showed no P uptake in the presence of nitrate when the sludge was highly diluted (ϳ0.005 g/liter); under these conditions, reduction of nitrate to nitrite did not occur, whereas P uptake by "Ca. Accumulibacter" clades occurred when nitrite was added. These results suggest that the "Ca. Accumulibacter" cells lack nitrate reduction capabilities and that P uptake by "Ca. Accumulibacter" is dependent upon nitrite generated by associated nitrate-reducing bacteria such as Dechloromonas and "Ca. Competibacter."

A Comparative Study of the Bacterial Community in Denitrifying and Traditional Enhanced Biological Phosphorus Removal Processes

“…5), which was attributed to its competitive advantage over Candidatus "Accumulibacter". Thus, our results supported and also extended previous findings (15,19). One clear advantage of our identification strategy was its independence on known PAO sequence information.…”

“…Based on the results of the above β-diversity analysis, the introduction of the A-A condition may have induced a shift in the bacterial community structure in EBPR, which has been reported in a previous FISH study (15).…”

“…Candidatus "Accumulibacter" may have also adapted to different ecological environments and exhibits various physiological properties similar to DPAO (5,11,12,15). In addition to the clades of Candidatus "Accumulibacter", Dechloromonasrelated bacteria had been detected in several EBPR processes operated under partially anoxic conditions (15,17,19). However, whether they function as PAO remains controversial.…”

“…According to Kong's study (19), a Dechloromonaslike organism (Bet135 FISH probe positive) was found to have a polyphosphate-accumulating phenotype by cultureindependent methods. Although another Dechloromonas-like organism (Dech453 FISH probe positive) was abundant in A-A-O SBR, it was predicted to only reduce nitrate to nitrite in the reactor, and Candidatus "Accumulibacter" acted as the real PAO in this system (15). Due to their wide distribution in wastewater treatment-related environments and disputed functions, the phosphorous removal activity of Dechloromonas-related bacteria needs to be elucidated in more detail.…”

“…Based on an elaborate online survey and subsequent phylogenetic analysis of reported 16S rRNA gene sequences from full scale wastewater treatment plants (WWTPs) and bench scale reactors (22,27), four clades (namely Acc-SG1, Acc-SG2, Acc-SG3 and Acc-SG4) were assigned by Kim et al (13). Candidatus "Accumulibacter" may have also adapted to different ecological environments and exhibits various physiological properties similar to DPAO (5,11,12,15). In addition to the clades of Candidatus "Accumulibacter", Dechloromonasrelated bacteria had been detected in several EBPR processes operated under partially anoxic conditions (15,17,19).…”