Biomass granulation in an aerobic:anaerobic-enhanced biological phosphorus removal process in a sequencing batch reactor with varying pH.

Long-term influences of different steady-state pH conditions on microbial community composition were determined by fluorescence in situ hybridization (FISH) in a laboratory scale reactor configured for enhanced biological phosphorus removal (EBPR). Chemical profiles were consistent with shifts in populations from polyphosphate-accumulating organisms (PAO) to glycogen-accumulating organisms (GAO) when pH fell from pH 7.5 to 7.0 and then to 6.5. While biomass was both dispersed and flocculated at pH 7.5, almost complete granulation occurred gradually after pH was dropped to 7.0, and these granules increased in size as the pH was reduced further to 6.5. Reverting back to pH 7.5 led to granule breakdown and corresponding increases in anaerobic phosphate release. Granules consisted almost entirely of Accumulibacter PAO cells, while putative GAO populations were always present in small numbers. Results suggest that low pH may contribute to granulation under these operational conditions. While chemical profiles suggested the PAO:GAO balance was changing as pH fell, FISH failed to reveal any marked corresponding increase in GAO abundances. Instead, TEM evidence suggested the Accumulibacter PAO phenotype was becoming more like that of a GAO. These data show how metabolically adaptable the Accumulibacter PAO can be under anaerobic:aerobic conditions in being able to cope with marked changes in plant conditions. They suggest that decreases in EBPR capacity may not necessarily reflect shifts in community composition, but in the existing population metabolism.

FACS enrichment and identification of floc-associated alphaproteobacterial tetrad-forming organisms in an activated sludge community.

A precise phylogenetic identity of the Defluviicoccus-related glycogen-accumulating organisms (GAO) observed after FISH probing in a novel activated sludge process removing phosphorus was sought with the aim of exploring the phylogenetic diversity of this important group. These organisms, whose sequences were not revealed in previously generated community wide 16S rRNA gene clone libraries, were identified using flow cytometry cell sorting of FISH-positive cells. Sequencing of a 16S rRNA gene clone library created from this sorted population identified the Defluviicoccus-related GAO as being highly related to previous identified GAO from enhanced biological phosphorus removal systems, despite a marked environmental difference between the two systems.

Changes in respiratory quinone profiles of enhanced biological phosphorus removal activated sludge under different influent phosphorus/carbon ratio conditions.

Changes in the microbial community of an enhanced biological phosphorus removal (EBPR) activated sludge system under different influent phosphorus/carbon (P/C) ratio conditions were investigated through evaluation of population respiratory quinone profiles. A total of 13 types of respiratory quinone homologs consisting of 3 types of ubiquinones (UQ) and 10 types of menaquinones (MK) were identified in this study. The dominant quinones were UQ-8 and MK-7 throughout the operational period. A higher P/C ratio (0.1) in the influent stimulated an increase in the mole fractions of UQ-8, MK-7, MK-8(H(4)), MK-9(H(4)) and MK-8(H(8)), suggesting that actinobacterial polyphosphate-accumulating organisms (PAO) containing partially hydrogenated MK, mainly MK-8(H(4)), were contributing to EBPR. However, when the P/C ratio gradually decreased from 0.1 to 0.01, the mole fractions of UQ-8 increased from 0.46 to 0.58, while MK-7, MK-8(H(2)), MK-8(H(4)), MK-9(H(4)), MK-8(H(8)) and MK-9(H(6)) markedly decreased. These changes in the respiratory quinone profiles suggest that glycogen-accumulating organisms corresponding to some Gammaproteobacteria had become dominant populations with a decrease in actinobacterial PAO. On the other hand, increasing abruptly the P/C ratio to 0.1 further caused an increase in the mole fraction of UQ-8, indicating that Rhodocyclus-related organisms were important PAO.

Characterization of denitrifying phosphate-accumulating organisms cultivated under different electron acceptor conditions using polymerase chain reaction-denaturing gradient gel electrophoresis assay.

To investigate the characteristics and the microbial diversity of denitrifying phosphate-accumulating organisms (DNPAOs) that are capable of conducting enhanced biological phosphorus removal (EBPR) using nitrate as electron acceptor, three sequencing batch reactors were operated under three different electron acceptor conditions, i.e., only oxygen, oxygen together with nitrate and only nitrate. Based on the chemical analysis concerning the biochemical transformation of each reactor, it was found that phosphate-accumulating organisms responsible for EBPR consisted of at least three populations including DNPAOs, and that the microbial community structure was changed according to the electron acceptor conditions. Also, the sludge cultivated with oxygen together with nitrate showed a drastic increase in the amount of phosphorus uptake under anoxic conditions, which suggested that a proportion of DNPAOs capable of utilizing nitrate under aerobic conditions were present. On the other hand, the change in microbial community structure depending on the type of electron acceptor was demonstrated by the analysis of the results of denaturing gradient gel electrophoresis of polymerase chain reaction-amplified 16S ribosomal DNA fragments. It was found that the bacteria commonly contained in all the reactors were Rhodocyclus sp. (96% identity) and Dechlorimonas sp. (97% identity) that belonged to the beta subclass of Proteobacteria on the basis of the analysis of the sequence excised from DGGE bands and the determination of phylogenetic affiliation. However, only the presence of Rhodocyclus sp. in all the reactors was demonstrated by fluorescent in situ hybridization analysis.