Cluster structure of anaerobic aggregates of an expanded granular sludge bed reactor

The metabolic properties and ultrastructure of mesophilic aggregates from a full-scale expanded granular sludge bed reactor treating brewery wastewate r are described. The aggregates had a very high methanogenic activity on ac etate (17.19 mmol of CH4/g of volatile suspended solids [VSS]-day or 1.1 g of CH4 chemical oxygen demand/g of VSS-day). Fluorescent in situ hybridizat ion using 16S rRNA probes of crushed granules showed that 70 and 30% of the cells belonged to the archaebacterial and eubacterial domains, respectivel y. The spherical aggregates were black but contained numerous whitish spots on their surfaces. Cross-sectioning these aggregates revealed that the whi te spots appeared to be white clusters embedded in a black matrix. The whit e clusters were found to develop simultaneously with the increase in diamet er. Energy-dispersed X-ray analysis and back-scattered electron microscopy showed that the whitish clusters contained mainly organic matter and no ino rganic calcium precipitates. The white clusters had a higher density than t he black matrix, as evidenced by the denser cell arrangement observed by hi gh-magnification electron microscopy and the significantly higher effective diffusion coefficient determined by nuclear magnetic resonance imaging. Hi gh-magnification electron microscopy indicated a segregation of acetate-uti lizing methanogens (Methanosaeta spp.) in the white clusters from syntrophi c species and hydrogenotrophic methanogens (Methanobacterium-like and Metha nospirillum-like organisms) in the black matrix. A number of physical and m icrobial ecology reasons for the observed structure are proposed, including the advantage of segregation for high-rate degradation of syntrophic subst rates.