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. 2010 Nov;76(22):7588-97.
doi: 10.1128/AEM.00864-10. Epub 2010 Sep 17.

Granule Formation Mechanisms Within an Aerobic Wastewater System for Phosphorus Removal

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Free PMC article

Granule Formation Mechanisms Within an Aerobic Wastewater System for Phosphorus Removal

Jeremy J Barr et al. Appl Environ Microbiol. .
Free PMC article

Abstract

Granular sludge is a novel alternative for the treatment of wastewater and offers numerous operational and economic advantages over conventional floccular-sludge systems. The majority of research on granular sludge has focused on optimization of engineering aspects relating to reactor operation with little emphasis on the fundamental microbiology. In this study, we hypothesize two novel mechanisms for granule formation as observed in three laboratory scale sequencing batch reactors operating for biological phosphorus removal and treating two different types of wastewater. During the initial stages of granulation, two distinct granule types (white and yellow) were distinguished within the mixed microbial population. White granules appeared as compact, smooth, dense aggregates dominated by 97.5% "Candidatus Accumulibacter phosphatis," and yellow granules appeared as loose, rough, irregular aggregates with a mixed microbial population of 12.3% "Candidatus Accumulibacter phosphatis" and 57.9% "Candidatus Competibacter phosphatis," among other bacteria. Microscopy showed white granules as homogeneous microbial aggregates and yellow granules as segregated, microcolony-like aggregates, with phylogenetic analysis suggesting that the granule types are likely not a result of strain-associated differences. The microbial community composition and arrangement suggest different formation mechanisms occur for each granule type. White granules are hypothesized to form by outgrowth from a single microcolony into a granule dominated by one bacterial type, while yellow granules are hypothesized to form via multiple microcolony aggregation into a microcolony-segregated granule with a mixed microbial population. Further understanding and application of these mechanisms and the associated microbial ecology may provide conceptual information benefiting start-up procedures for full-scale granular-sludge reactors.

Figures

FIG. 1.
FIG. 1.
EBPR performance (a) and particle size (b) of the Synthetic-1 SBR treating synthetic wastewater over a 168-day period. The vertical lines indicate where cycle changes were made to remove floccular material from the system and where the influent phosphorus concentration was halved due to an increase in the reactor volume exchange ratio; however, the total phosphorus treated did not change (see Materials and Methods). The “initial granulation” arrows indicate when the 50th-percentile particle size was above 200 μm, indicative of initial granule formation. The “mature granulation” arrows indicate when large, developed granules were present. The “fully granulated” arrows indicate when the majority of flocs were removed from the system. The 10th percentile indicates that 10% of particles were below this size distribution, the 50th percentile is the average (or median) particle size distribution, and the 90th percentile indicates 10% of the particles were above this size distribution.
FIG. 2.
FIG. 2.
Stereomicroscope images of white (a), yellow (b), and off-white (c) granules and light microscope images of white (d), yellow (e), and off-white (f) granules from the Synthetic-1 reactor. The images of white and yellow granules were taken on day 63 of Synthetic-1 operation, and the images of off-white granules were taken on day 133 of Synthetic-1 operation. White and yellow granules were present in the reactor only during the initial stages of granulation, after which a single homogeneous off-white granule population developed. All scale bars, 500 μm.
FIG. 3.
FIG. 3.
CLSM image of cryosectioned granules after FISH, with “Candidatus Accumulibacter phosphatis” in blue, “Candidatus Competibacter phosphatis” in yellow, and all other bacteria in green. (a) Synthetic-1 white granules were imaged on day 98 of reactor operation and were dominated by “Candidatus Accumulibacter phosphatis” with a solid, compact outer-wall structure. (b) Synthetic-1 yellow granules were imaged on day 98 of reactor operation and had a diverse bacterial population, were generally larger, and consisted of numerous compartmentalized microcolony structures. (c) Synthetic-1 off-white granules were imaged on day 168 of reactor operation and appeared as a mixture of the white and yellow granules with both solid outer walls and microcolony structures. (d) Domestic white granules were imaged on day 68 of reactor operation, were dominated by “Candidatus Accumulibacter phosphatis,” and exhibited a solid, compact outer-wall structure similar to that of the Synethic-1 white granules. (e) Domestic yellow granules were imaged on day 68 of reactor operation and exhibited a solid structure with fewer microcolonies and a more diverse bacterial population than the Synthetic-1 yellow granules. All scale bars, 100 μm.
FIG. 4.
FIG. 4.
TEM image of an outer edge of a white granule (a) and a yellow granule (b) taken on day 98 of Synthetic-1 reactor operation. White granules were dominated by one cellular morphotype, likely “Candidatus Accumulibacter phosphatis,” in a solid homogeneous layer. Yellow granules showed diverse numbers of bacterial morphotypes growing in distinct microcolony structures. All scale bars, 10 μm.
FIG. 5.
FIG. 5.
Unrooted abbreviated phylogenetic tree obtained by the neighbor-joining method, showing the positions of four ppkI clones from white (W) and yellow (Y) granules with known ppkI gene sequences from both the type I and type II “Candidatus Accumulibacter phosphatis” lineages. All cloned sequences were positioned within the type I clade branch. GenBank sequence accession numbers are given in parentheses. The numbers at the nodes show the percentage bootstrap values. The scale bar indicates the number of changes per site.
FIG. 6.
FIG. 6.
Stereomicroscopy and homogenized FISH images of white and yellow granules from the Synthetic-2 reactor (a to d) on day 182 of operation and the Domestic reactor (e to h) on day 68 of operation. The FISH images show “Candidatus Accumulibacter phosphatis” in blue, “Candidatus Competibacter phosphatis” in yellow, and all other bacteria in green. Scale bars, 200 μm (a and b), 500 μm (e and f), and 20 μm (c, d, g, and h).
FIG. 7.
FIG. 7.
16S rRNA gene tRFLP cluster analysis using the Domestic reactor samples. Three white (W) and three yellow (Y) PCR products were individually digested with 3 different restriction endonucleases, AluI (a), MspI (b), and Sau3AI (c). Cluster analysis of the resulting tRFLP electrograms showed samples from the same granule “type” grouped together. The Euclidean distance scale is shown for the cluster analysis, and the tRFLP fragment length (DNA base number) is indicated by a scale bar above each electrogram.
FIG. 8.
FIG. 8.
Two hypothesized mechanisms for granulation. (a) Microcolony outgrowth (white granules), where a small microcolony composed of one bacterial type is selected for and grows outward, eventually forming its own dense homogeneous granule. (b) Microcolony aggregation (yellow granules), where numerous small microcolonies composed of different bacterial types aggregate and grow as an entity, eventually forming a rough, highly segregated granule. (Courtesy of Frances Slater.)

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