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Applied and Environmental Microbiology, September 2002, p. 4629-4636, Vol. 68, No. 9
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.9.4629-4636.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Abundance and Phylogenetic Affiliation of Iron Reducers in Activated Sludge as Assessed by Fluorescence In Situ Hybridization and Microautoradiography

Jeppe Lund Nielsen,1 Stefan Juretschko,2 Michael Wagner,2 and Per Halkjær Nielsen1*

Department of Environmental Engineering, Aalborg University, DK-9000 Aalborg, Denmark,1 Lehrstuhl für Mikrobiologie der Technischen Universität München, D-85350 Freising, Germany2

Received 8 March 2002/ Accepted 25 June 2002

Microautoradiography (MAR) was used to enumerate acetate-consuming bacteria under Fe(III)-reducing conditions in activated sludge. This population is believed to consist of dissimilatory iron-reducing bacteria, because the applied incubation conditions and the use of specific inhibitors excluded consumption of radiolabeled acetate by other physiological groups such as sulfate reducers. By use of this approach, dissimilatory iron reducers were found in a concentration of 1.1 x 108 cells per ml, corresponding to approximately 3% of the total cell count as determined by DAPI (4',6'-diamino-2-phenylindoledihydrochloride-dilactate) staining. The MAR enumeration method was compared to the traditional most-probable-number (MPN) method (FeOOH-MPN) and a modified MPN method, which contains Ferrozine directly within the MPN dilutions to determine the production of small amounts of ferrous iron (Ferrozine-MPN). The Ferrozine-MPN method yielded values 6 to 10 times higher than those obtained by the FeOOH-MPN method. Nevertheless, the MAR approach yielded counts that were 100 to 1,000 times higher than those obtained by the Ferrozine-MPN method. Specific in situ Fe(III) reduction rates per cell (enumerated by the MAR method) were calculated and found to be comparable to the respective rates for pure cultures of dissimilatory iron-reducing bacteria, suggesting that the new MAR method is most reliable. A combination of MAR and fluorescence in situ hybridization was used for phylogenetic characterization of the putative iron-reducing bacteria. All activated-sludge cells able to consume acetate under iron-reducing conditions were targeted by the bacterial oligonucleotide probe EUB338. Around 20% were identified as gamma Proteobacteria, and 10% were assigned to the delta subclass of Proteobacteria.

* Corresponding author. Mailing address: Department of Environmental Engineering, Aalborg University, Sohngaardsholmsvej 57, DK-9000 Aalborg, Denmark. Phone: 45 96358505. Fax: 45 98142555. E-mail: phn{at}civil.auc.dk.

Applied and Environmental Microbiology, September 2002, p. 4629-4636, Vol. 68, No. 9
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.9.4629-4636.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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