TetraSpec Fluorescent Microsphere Standards (Molecular Probes, OR, USA) detected channels and pores with diameters greater than 0.1 |xm. All were visualized with Fluoview300 confocal laser scanning microscope (CLSM) (Olympus, Japan) as described previously (Tay et al., 2002a,b). Observations with CLSM at 1000 x magnification showed that the beads did not adhere to the cell surface. Therefore, their distribution within the granule is not a measure of the adsorption of the beads onto the granule matrix, but indicates the penetration of the beads into the granule interior by passage through pore and channel structures which have to be larger than 0.1 |xm in size. The incubation period of 4 h was more than sufficient to allow complete penetration of the beads into the granule interior. Test measurements performed using different incubation times showed that bead penetration reached saturation levels within 1 h of incubation.
Mass transfer rate in microbial aggregates may be enhanced by the formation of channels and pores that interconnect the surface and the interior. Such channels and pores had been previously observed in aerobic biofilms (Massol-Deya et al., 1995). The aerobic granules in this current study also contain channels and pores that penetrated to depths of up to 900 |xm from the surface of the granule (Fig. 6.4d). Channels and pores were detected in the granule and porosity values peaked at depths of 300-500 |xm from the granule surface (Fig. 6.4d). The thickness of the porous layer in the granule positively correlated with the granule diameter. For example, a granule with a diameter of 550 |xm had a porous layer with a thickness of 250 |xm, and a granule with a diameter of 1000 |xm had a porous layer with a thickness of 350 |xm. The biomass and the porosity profiles were also observed to drop at the same depth below the granule surface. There was no penetration of 0.1 |xm microspheres to the central core of the large granules (Ivanov et al., 2004).
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