While granulated activated sludge exhibits high productivity, the processes of granule formation are incompletely studied. The processes of granule formation and succession of communities were investigated in a laboratory sequencing batch reactor (SBR) under conditions for enhanced biological phosphorus removal (EBPR) using microbiological and molecular techniques. Active consumption of acetate, primarily by the phosphate-accumulating organisms (PAO), commenced at day 150 of cultivation. This was indicated by the high ratio of molar P-released/acetate uptake (0.73–0.77 P-mol/C-mol), characteristic of PAO. During this period, two types of granule-like aggregates formed spontaneously out of the activated sludge flocs. The aggregates differed in morphology and microbial taxonomic composition. While both aggregate types contained phosphorus-enriched bacterial cells, PAO prevailed in those of morphotype I, and glycogen-accumulating organisms (GAOs) were predominant in the aggregates of morphotype II. After 250 days, the elimination of the morphotype II aggregates from the reactor was observed. The subsequent selection of the community was associated with the development of the morphotype I aggregates, in which the relative abundance of PAO increased significantly, resulting in higher efficiency of phosphorus removal. Metagenomic analysis revealed a predominance of the organisms closely related toCandidatusAccumulibacter IС and IIС and ofCa.Accumulibacter IIB among the PAO. Based on the content of the genes of the key metabolic pathways, the genomes of potential PAO belonging to the generaAmaricoccus, Azonexus, Thauera, Zoogloea, Pinisolibacter, andSiculibacilluswere selected. The patterns of physicochemical processes and the microbiome structure associated with granule formation and succession of the microbial communities were revealed.

Formation of a community of phosphate-accumulating microorganisms in a laboratory sequential batch reactor (SBR) ensuring alternated aerobic and anaerobic conditions during periodic removal and addition of the medium were investigated. The bioreactor removed 50% phosphorus from the incoming medium after 22 days from the start-up. Microscopy and X-ray microassay revealed the of cells of diverse morphology that contained phosphorus-enriched granules. High-throughput sequencing of the 16S rRNA gene fragments carried out on days 0, 8, 15, and 22 showed changes in the community composition and its decreasing diversity. On day 22, approximately twofold increase of the relative abundances of Bacteroidetes (up to 43% of the 16S rRNA gene sequences) and Proteobacteria of the classes alpha (up to 15%) and beta (up to 27%) was observed. While at the onset of the reactor operation, typical PAOs related to “Candidatus Accumulibacter” (class Betaproteobacteria) constituted 0.2% of the community, they were not detected on day 22. The most likely PAO candidates were beta-proteobacteria of the genus Dechloromonas, the share of which increased from 0.7 to 11% by the time of the highest phosphorus removal from the inflowing medium. The relative abundance of heterotrophs of the genus Zoogloea (family Rhodocyclaceae) increased from 0.1 to 11.5%.

A microbial community enriched with phosphate-accumulating organisms (PAO) was obtained in a laboratory sequencing batch reactor after 150 days of cultivation. Analysis of phosphate dynamics in the medium and the results of Raman scattering spectroscopy indicated the cycle of phosphate consumption and release during cultivation under oxic and anoxic conditions, respectively, which was typical of PAO. The highest content of intracellular phosphorus was 16.5 ± 0.15% of the dry ash-free biomass. Molecular genetic analysis and FISH revealed the taxonomic diversity of the microbial community, in which members of the “Ca. Аccumulibacter” group were the major PAO.

Candidatus Accumulibacter belongs to phosphate-accumulating organisms (PAOs) which exhibit a cyclic metabolism and are capable of intracellular polyphosphate accumulation and their hydrolysis under feast-famine anaerobic-aerobic cycling. In consortia of activated sludge microorganisms, these bacteria are responsible for enhanced biological phosphorus removal (EBPR). The spectrum of the substrates used by Ca. Accumulibacter remains insufficiently studied. It was investigated by measuring the oxygen uptake rates (OUR) of Ca. Accumulibacter-enriched culture supplemented with 17 different organic substrates. The highest oxygen uptake rate values were observed in the presence of tryptone, volatile fatty acids (acetate, propionate, and butyrate), succinate, pyruvate, and amino acids (aspartate and glutamate). Phosphate dynamics in the medium under shifts from anaerobic to aerobic cultivation in batch experiments were studied for these compounds (except for tryptone). All tested substrates were shown to cause phosphate cycling (release in the anaerobic phase and uptake in the aerobic one), with OURs for the substrates correlating with the number of phosphates consumed during the aerobic phase. It was concluded that OUR may be used as an indicator of the monosubstrates used by Ca. Accumulibacter in the anaerobic/aerobic cycle. The possible pathways for substrate transport and metabolism by Ca. Accumulibacter are discussed using stoichiometric data and the results of metagenomic analysis.

Ability of the aerobic PAO community enriched with Ca. Accumulibacter to use nitrate and nitrite as electron acceptors was investigated. Despite the presence of the genes related to denitrification, it was shown that replacement of oxygen by nitrate resulted in a loss of the PAO phenotype. This could be explained by the inability of Ca. Accumulibacter, which predominated in the bioreactor, to carry out the first stage of denitrification. Using nitrite in an acute experiment resulted in a noticeable cycling of phosphate concentrations in the bioreactor. Phosphate consumption during the anoxic period, however, was considerably less than under conditions of the aerobic cycle. It was concluded that Ca. Accumulibacter could use nitrite as an alternative elector acceptor, albeit with significantly lower phosphorus removal efficiency compared to this process in the presence of oxygen.