Comparison of the bacterial community composition in the granular and the suspended phase of sequencing batch reactors

Abstract

Granulation of activated sludge is an increasingly important area within the field of wastewater treatment. Granulation is usually achieved by high hydraulic selection pressure, which results in the wash-out of slow settling particles. The effect of the harsh wash-out conditions on the granular sludge ecosystem is not yet fully understood, but different bacterial groups may be affected to varying degrees. In this study, we used high-throughput amplicon sequencing to follow the community composition in granular sludge reactors for 12 weeks, both in the granular phase and the suspended phase (effluent). The microbiome of the washed out biomass was similar but not identical to the microbiome of the granular biomass. Certain taxa (e.g. Flavobacterium spp. and Bdellovibrio spp.) had significantly (p < 0.05) higher relative abundance in the granules compared to the effluent. Fluorescence in situ hybridization images indicated that these taxa were mainly located in the interior of granules and therefore protected from erosion. Other taxa (e.g. Meganema sp. and Zooglea sp.) had significantly lower relative abundance in the granules compared to the effluent, and appeared to be mainly located on the surface of granules and therefore subject to erosion. Despite being washed out, these taxa were among the most abundant members of the granular sludge communities and were likely growing fast in the reactors. The ratio between relative abundance in the granular biomass and in the effluent did not predict temporal variation of the taxa in the reactors, but it did appear to predict the spatial location of the taxa in the granules.

Keywords: Aerobic granular sludge; Microbial community composition; Sequencing batch reactors; Spatial distribution; Temporal variation; Wash-out dynamics.

Fig. 1

Non-metric multidimensional scaling (NMDS) ordination based on Bray–Curtis dissimilarity matrix. Samples are indicated with the number of days since seeding. R1E, R2E, R3E: effluent samples (suspended phase); R1R, R2R, R3R: samples from the reactor content (granular phase), SS seed sludge

Fig. 2

Average relative read abundance (n = 13) of the most abundant genera common for all six sample categories. These 19 genera add up to 65 ± 11% of the total population in the samples. R1E, R2E, R3E: effluent samples (suspended phase); R1R, R2R, R3R: samples from the reactor content (granular phase)

Fig. 3

Boxplots of the retention ratios during steady-state operation (weeks 7–12) in R1 (a), R2 (b) and R3 (c). Values significantly different from 1 are marked with asterisk (p values are shown above the plots)

Fig. 4

FISH-CLSM images of selected bacteria with retention ratios significantly lower than one. Cryosections of granules at ×200 magnification and detailed sections at ×400 magnification. a, b Meganema perideroedes in granules from R2; c Zoogloea spp. in granules from R2; d Zoogloea spp. in aerobic granules from R3. Grey total cells (Syto 40); red Meganema perideroedes (a, b) and Zoogloea spp. (c, d)

Fig. 5

FISH-CLSM images of selected bacteria with retention ratios significantly higher than one. Cryosections of granules at ×200 magnification and detailed sections at ×400 magnification. a, b Genus Bdellovibrio in granules from R1; c, d Flavobacterium spp. in granules from R1. Grey total cells (Syto 40); red genus Bdellovibrio (a, b) and Flavobacterium spp. (c, d)