The deterioration of the granules was studied by labeling cells with 1 |xg L-1 of fluorescent lipophilic tracer DiIC18(3) (Molecular Probes, OR, USA). The tracer was readily taken up by the cells. The in-solution concentration of the tracer one day after its introduction into the reactor was less than 1% of the concentration detected in a suspension of particles produced by disintegrating granules in a 2 mL tube with phosphate-buffered saline (PBS) using a Mini-Beadbeater (Biospec Products, Inc., Bartlesville, OK, USA) for 100 s at 500 rpm. The amount of fluorescence due to the lipophilic stain was determined using a Luminescence Spectrometer LS-50B (Perkin-Elmer, Boston, MA 02118, USA). Background due to autofluorescence of biomass was excluded from the reported values.
The granules are not only able to degrade organic matter but are also able to remove nano- and microparticles from wastewater due to microchannels and pores in the matrix of the granules. To detect the removal of 0.1 |xm, 0.6 |xm, 4.2 |xm fluorescent microspheres, and cells of Escherichia coli, stained by permeable nucleic acid stain SYTO9™, the granules were incubated with these particles. Total number of the particles bigger than 0.1 |xm in the reactors was approximately 4 x 107per mL, and 23% of these particles were bacterial cells. The cells of Escherichia coli and 4.2 |xm microbeads were accumulated within 250 |xm in the upper layer of the microbial granule but small 0.1 |xm microbeads penetrated to the depth approximately 500 |xm in the granules (Fig. 6.5).
Microbial granules contained also attached ciliates (Fig. 6.2d) but accumulation of the particles in protozoan cells was smaller than in the granule matrix. Kinetics of particle sorption was revealed by flow cytometry and fluorescence spectrometry. Almost half of the stained cells of E. coli can be removed by the granules for one hour. The ability of the microbial granules to remove the particles can enhance their function in aerobic treatment of wastewater.
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