Radial Structures in Granule

Important structural property of microbial granules, related to their bioengineering functions, is arrangement of granule components as radial sub-aggregates, spherical sub-granules, and concentric layers (Fig. 6.3). Depth, thickness, and arrangement of these components can affect the formation, stability, and activity of the granules. The sub-aggregates inside the granules may be arranged randomly, in a radial direction (Fig. 6.3a), or as concentric layers (Fig. 6.3c). Larger granules may also result from merging of smaller granules.

Radial aggregates Concentric layer

Fig. 6.3. Different microaggregates of cell aggregates (a) radially arranged microaggregates of ammonia-oxidizing bacteria (bright structures) in a 3D image produced by CLSM of the granule; (b) biofilm of nitrifying bacteria on the surface of Noble Agar with oxygen supply through the agar surface and ammonia supply from the agar bottom; one layer was a uniform biofilm but another one contained aggregates of nitrifying bacteria arranged perpendicular to the agar surface; (c) concentric layer of Bacteroides spp.

Radial aggregates Concentric layer

Fig. 6.3. Different microaggregates of cell aggregates (a) radially arranged microaggregates of ammonia-oxidizing bacteria (bright structures) in a 3D image produced by CLSM of the granule; (b) biofilm of nitrifying bacteria on the surface of Noble Agar with oxygen supply through the agar surface and ammonia supply from the agar bottom; one layer was a uniform biofilm but another one contained aggregates of nitrifying bacteria arranged perpendicular to the agar surface; (c) concentric layer of Bacteroides spp.

In spherical granular biofilm, ammonia-oxidizing bacteria were arranged in radially elongated aggregates within a layer with a depth from 70 to 100 |xm from the surface of the biofilm (Tay et al. 2002a; Ivanov et al., 2005a,b) as shown in Figs 6.3a and 6.4b. Labeled oligonucleotide probes Bacto1080 and Nsm156 probes were applied using the FISH procedure.

Formation of radial aggregates of nitrifying bacteria (Figs 6.3a and 6.4b), in a direction that is normal to the granule surface, is probably driven by the co-existence of steep oxygen gradient and reverse ammonia gradient created by release of ammonia from the central core of granule where biomass is lyzed. The hypothesis on reverse gradient of ammonia in the granules was examined in independent experiments during the growth of enrichment culture of nitrifying bacteria in Noble Agar where oxygen was supplied through the agar surface but where ammonia was supplied from the bottom of the agar layer. Ammonia-oxidizing bacteria formed two layers. The first layer was a uniform biofilm but the second layer contained aggregates of nitrifying bacteria aligned normal to the agar surface (Fig. 6.3b). The decreasing widths of these nitrifying aggregates probably reflect the dependence of growth rate on the available concentration of dissolved oxygen and ammonia.

Cells of ammonia-oxidizing bacteria are often arranged in the laminar biofilms as microbial colonies embedded in slime attached to a carrier surface (Okabe et al., 1999). In laminar microbial biofilm on sea shells ammonia-oxidizing cells were arranged as a layer of vertically elongated aggregates (Ivanov et al., 2005b). These aggregates were embedded within the matrix formed by other bacteria. Vertically elongated aggregates seemed to be capable of multiplication due to their lateral growth and further splitting. Vertical (radial) cell aggregates may be ecologically important in bacterial biofilms because they have a higher surface-to-volume ratio (S/V) than laminated biofilms. For example, S/Vfor a 100 |xm layer of biofilm is 0.01 |xm2/^,m3. However, if the microbial layer consists of vertical 20-^m-diameter cylinders, arranged so that the axes of neighboring cylinders are 40 |xm apart, then S/V = 0.21. Therefore, when the microbial biofilm is arranged as a layer of vertical aggregates, the S/V ratio, and respectively, the rates of substrate transfer, microbial metabolism and growth, could be 20 times higher than the same parameters for laminated biofilms.

Vertically arranged, pear-shaped aggregates of ammonia-oxidizing bacteria have been shown in spherical suspended microbial biofilms

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Fig. 6.4. Layers, channels, and pores in aerobic granules (a) shows an edge of the granule by evaluating the decrease of the light intensity through the granule; (b) shows a layer of aerobic, ammonia-oxidizing bacteria Nitrosomonas spp; (c) shows the distribution of microbial biomass (by measuring fluorescence intensity of hybridized Eub338 probe) (curve 1) and a layer of anaerobic bacteria Bacteroides spp. (curve 2); (d) shows the presence of the channels and the pores in the granule by measuring fluorescence of 0.1 ^m microspheres penetrating into the granules from the medium; (e) shows the fluorescence of dead cells; (f) shows the fluorescence of polysaccharides stained by FITC-labeled ConA. The dotted line in all the figures represent the granule surface. The arrow in all the figures represent the granule center.

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Fig. 6.4. Layers, channels, and pores in aerobic granules (a) shows an edge of the granule by evaluating the decrease of the light intensity through the granule; (b) shows a layer of aerobic, ammonia-oxidizing bacteria Nitrosomonas spp; (c) shows the distribution of microbial biomass (by measuring fluorescence intensity of hybridized Eub338 probe) (curve 1) and a layer of anaerobic bacteria Bacteroides spp. (curve 2); (d) shows the presence of the channels and the pores in the granule by measuring fluorescence of 0.1 ^m microspheres penetrating into the granules from the medium; (e) shows the fluorescence of dead cells; (f) shows the fluorescence of polysaccharides stained by FITC-labeled ConA. The dotted line in all the figures represent the granule surface. The arrow in all the figures represent the granule center.

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(Ivanov et al., 2005b). It is likely that more examples of such vertically arranged aggregates in microbial biofilm could be found. Presence of these vertically arranged microbial aggregates must be taken into account in the models of microbial biofilms (Morgenroth et al., 2004; Picioreanu et al., 2004) and in the mathematical model of aerobic microbial granule.

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