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Applied and Environmental Microbiology, June 2003, p. 3626-3635, Vol. 69, No. 6
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.6.3626-3635.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Characterization of an Autotrophic Nitrogen-Removing Biofilm from a Highly Loaded Lab-Scale Rotating Biological Contactor

Kris Pynaert,¹ Barth F. Smets,^1, Stijn Wyffels,² Daan Beheydt,¹ Steven D. Siciliano,^1, and Willy Verstraete^1*

Laboratory of Microbial Ecology and Technology,¹ Laboratory for Applied Physical Chemistry, Faculty of Agricultural and Applied Biological Sciences, Ghent University, B-9000 Ghent, Belgium²

Received 8 November 2002/ Accepted 26 February 2003

In this study, a lab-scale rotating biological contactor (RBC) treating a synthetic NH₄⁺ wastewater devoid of organic carbon and showing high N losses was examined for several important physiological and microbial characteristics. The RBC biofilm removed 89% ± 5% of the influent N at the highest surface load of approximately 8.3 g of N m^-2 day^-1, with N₂ as the main end product. In batch tests, the RBC biomass showed good aerobic and anoxic ammonium oxidation (147.8 ± 7.6 and 76.5 ± 6.4 mg of NH₄⁺-N g of volatile suspended solids [VSS]^-1 day^-1, respectively) and almost no nitrite oxidation (< 1 mg of N g of VSS^-1 day^-1). The diversity of aerobic ammonia-oxidizing bacteria (AAOB) and planctomycetes in the biofilm was characterized by cloning and sequencing of PCR-amplified partial 16S rRNA genes. Phylogenetic analysis of the clones revealed that the AAOB community was fairly homogeneous and was dominated by Nitrosomonas-like species. Close relatives of the known anaerobic ammonia-oxidizing bacterium (AnAOB) Kuenenia stuttgartiensis dominated the planctomycete community and were most probably responsible for anoxic ammonium oxidation in the RBC. Use of a less specific planctomycete primer set, not amplifying the AnAOB, showed a high diversity among other planctomycetes, with representatives of all known groups present in the biofilm. The spatial organization of the biofilm was characterized using fluorescence in situ hybridization (FISH) with confocal scanning laser microscopy (CSLM). The latter showed that AAOB occurred side by side with putative AnAOB (cells hybridizing with probe PLA46 and AMX820/KST1275) throughout the biofilm, while other planctomycetes hybridizing with probe PLA886 (not detecting the known AnAOB) were present as very conspicuous spherical structures. This study reveals that long-term operation of a lab-scale RBC on a synthetic NH₄⁺ wastewater devoid of organic carbon yields a stable biofilm in which two bacterial groups, thought to be jointly responsible for the high autotrophic N removal, occur side by side throughout the biofilm.

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* Corresponding author. Mailing address: Laboratory of Microbial Ecology and Technology (LabMET), Faculty of Agricultural and Applied Biological Sciences, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium. Phone: 32 (0)9 264 59 76. Fax: 32 (0)9 264 62 48. E-mail: willy.verstraete{at}rug.ac.be.

Present address: Environmental Engineering and Microbiology Programs, University of Connecticut, Storrs, CT 06269-2037.

Present address: Department of Soil Science, University of Saskatchewan, Saskatoon, SK S7N5A8, Canada.

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Applied and Environmental Microbiology, June 2003, p. 3626-3635, Vol. 69, No. 6
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.6.3626-3635.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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