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Applied and Environmental Microbiology, August 2001, p. 3683-3692, Vol. 67, No. 8
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.8.3683-3692.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

G. Gonzalez-Gil,1,* P. N. L. Lens,1,2 A. Van Aelst,3 H. Van As,2 A. I. Versprille,4 and G. Lettinga1

Sub-department of Environmental Technology,1 Department of Molecular Physics,2 and Department of Plant Cytology and Morphology,3 University of Wageningen, 6700 EV Wageningen, and Biothane Systems International, 2600 GB Delft,4 The Netherlands

Received 18 January 2001/Accepted 4 May 2001

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 CH4/g of volatile suspended solids [VSS]·day or 1.1 g of CH4 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.

* Corresponding author. Mailing address: Sub-department of Environmental Technology, University of Wageningen, Bomenweg 2, P.O. Box 8129, 6700 EV Wageningen, The Netherlands. Phone: 31 (0) 317 485595. Fax: 31 (0) 317 482108. E-mail: graciela.gonzalez{at}algemeen.mt.wau.nl.

Applied and Environmental Microbiology, August 2001, p. 3683-3692, Vol. 67, No. 8
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.8.3683-3692.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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