Cluster Structure of Anaerobic Aggregates of an Expanded Granular Sludge Bed Reactor

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Abstract
The metabolic properties and ultrastructure of mesophilic aggregates from a full-scale expanded granular sludge bed reactor treating brewery wastewater are described. The aggregates had a very high methanogenic activity on acetate (17.19 mmol of CH 4 /g of volatile suspended solids [VSS]·day or 1.1 g of CH 4 chemical oxygen demand/g of VSS·day). Fluorescent in situ hybridization using 16S rRNA probes of crushed granules showed that 70 and 30% of the cells belonged to the archaebacterial and eubacterial domains, respectively. The spherical aggregates were black but contained numerous whitish spots on their surfaces. Cross-sectioning these aggregates revealed that the white spots appeared to be white clusters embedded in a black matrix. The white clusters were found to develop simultaneously with the increase in diameter. Energy-dispersed X-ray analysis and back-scattered electron microscopy showed that the whitish clusters contained mainly organic matter and no inorganic calcium precipitates. The white clusters had a higher density than the black matrix, as evidenced by the denser cell arrangement observed by high-magnification electron microscopy and the significantly higher effective diffusion coefficient determined by nuclear magnetic resonance imaging. High-magnification electron microscopy indicated a segregation of acetate-utilizing methanogens ( Methanosaeta spp.) in the white clusters from syntrophic species and hydrogenotrophic methanogens ( Methanobacterium -like and Methanospirillum -like organisms) in the black matrix. A number of physical and microbial ecology reasons for the observed structure are proposed, including the advantage of segregation for high-rate degradation of syntrophic substrates.