Aerobic granules: microbial landscape and architecture, stages, and practical implications

Abstract

For the successful application of aerobic granules in wastewater treatment, granules containing an appropriate microbial assembly able to remove contaminants should be retained and propagated within the reactor. To manipulate and/or optimize this process, a good understanding of the formation and dynamic architecture of the granules is desirable. Models of granules often assume a spherical shape with an outer layer and an inner core, but limited information is available regarding the extent of deviations from such assumptions. We report on new imaging approaches to gain detailed insights into the structural characteristics of aerobic granules. Our approach stained all components of the granule to obtain a high quality contrast in the images; hence limitations due to thresholding in the image analysis were overcome. A three-dimensional reconstruction of the granular structure was obtained that revealed the mesoscopic impression of the cavernlike interior of the structure, showing channels and dead-end paths in detail. In "old" granules, large cavities allowed for the irrigation and growth of dense microbial colonies along the path of the channels. Hence, in some areas, paradoxically higher biomass content was observed in the inner part of the granule compared to the outer part. Microbial clusters "rooting" from the interior of the mature granule structure indicate that granules mainly grow via biomass outgrowth and not by aggregation of small particles. We identify and discuss phenomena contributing to the life cycle of aerobic granules. With our approach, volumetric tetrahedral grids are generated that may be used to validate complex models of granule formation.

FIG 1

(A) Macroimpression of aerobic granular sludge. (B) Granules grown in propionate and sampled at day 169. (C) Stereomicrograph of granules having different “life” stage (see the text). (D) Low-magnification SEM image of a granule showing large clusters. (E) SEM image of the surface of a granule. (F) Zoom image of panel E reveals some filamentous bacteria sheltered between clusters. (G) Detailed impression of the surface of a cluster.

FIG 2

Thin section images of a propionate-cultivated granule sampled at day 169. (A) Fluorescence image showing large clusters of Accumulibacter-like bacteria in pink. Competibacter-like bacteria are shown in cyan and other microorganisms are shown in blue. (B) Zoom image corresponding to the box in panel A showing growing clusters. (C and D) Bright-field and dark-field images, respectively, of a contiguous section to that in panel A and stained with H&E. The arrows in panel C indicate glue-like structures.

FIG 3

Thin section images of an acetate-cultivated granule sampled at day 169. (A) Fluorescence image showing clusters of Accumulibacter-like bacteria in pink. Competibacter-like bacteria are shown in cyan and other microorganisms are shown in blue. (B) Zoom image corresponding to the box in panel A. (C and D) Bright-field and dark-field images, respectively, of a contiguous section to that in panel A and stained with H&E showing the outgrow structure of the clusters.

FIG 4

Bright-field microimages from thin sections obtained from the center of granules and stained with H&E. The “bl” refers to bulk liquid. (A to C) Images show that various types of dense microcolonies (dark pink/purple) are embedded in a matrix of less dense cellular material (light pink). (D) Overview of an entire granule showing a highly irrigated inner structure. (E) Zoom image corresponding to the inset square in panel D showing colonies irrigated by the inner channels.

FIG 5

(A and B) Bright-field images, respectively, showing clusters of biomass that “root” or outgrow from the granule. The scale bar in panel B applies similarly to panel A. (C and D) Dark-field images showing similar shapes of outgrow of clusters observed in other granules. The arrows indicate the start of outgrows. The center of the granules is opposite to the arrows.

FIG 6

(A) Fractal dimension estimated for various types of granules. For comparisons, microscopic images of anaerobic granules previously reported (29) were used. (B) Fractal dimension as a function of distance from the granule center.

FIG 7

Internal porosity as a function of distance from the center of the granule.

FIG 8

(A) 3D rendering of an aerobic granule. (B) Volumetric representation of half of a granule as tetrahedral grid. For this reconstruction, 41 slices of 12 μm each (see Fig. S2 in the supplemental material) were used.