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Use of the ammonia-oxidizing bacterial-specific phylogenetic probe Nso1225 as a primer for fingerprint analysis of ammonia-oxidizer communities

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Abstract

Autotrophic ammonia-oxidizing bacteria (AOB) are an essential component of nitrifying wastewater treatment systems. The molecular tools used in group-specific studies are mostly based on the sequence of the 16S rRNA gene, but they have not proved to be fully specific. In this study, the sequence of the FISH probe Nso1225R was used as a reverse primer in order to analyze the AOB composition of several environmental samples by denaturing gradient gel electrophoresis (DGGE). For this purpose, samples from several environments, including aerated reactors, water treatment wetlands, and pilot plants, both aerobic and anaerobic, were analyzed. PCR fragments displayed a DGGE pattern consisting of bands melting between 30 and 40% denaturant, and a series of unresolved bands above 45%, mostly corresponding to AOB and β-non-AOB, respectively. This second set of bands corresponded to environments subjected to severe oxygen restrictions. AOB sequences showed similarity percentages higher than 92% with those of known β-AOB. Nso1225R, therefore, proved to be a good molecular phylogenetic marker for AOB samples from well-aerated systems, showing a higher specificity than the group-specific primers used previously.

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Fig. 1A–K.
Fig. 2.

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Abbreviations

AOB :

Ammonia-oxidizing bacteria

DGGE :

Denaturing gradient gel electrophoresis

MOB :

Methane-oxidizing bacteria

Nc. :

Nitrosococcus

Nm. :

Nitrosomonas

Ns.:

Nitrosospira

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Acknowledgements

We thank Dr. Annette Bollmann (Netherlands Institute of Ecology, Center for Limnology, Maarsen) and Dr. Janne B. Utåker (Laboratory of Microbial Gene Technology, Dept. of Chemistry and Biotechnology Agricultural University of Norway) for kindly providing the genomic DNA of ammonia oxidizing bacteria. We would also like to thank the Laboratory of Chemical and Environmental Engineering from the University of Girona for providing the samples from the biodiscs, the aerobic and anaerobic reactors used in this work. This research was funded by the Spanish Ministry of Science and Technology (ref. CICYT REN2000 and BIO99-1014-CO2-01). Laia Calvó is the recipient of a fellowship from the Spanish Ministry of Science and Technology.

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  1. Laboratory of Molecular Microbial Ecology, Institute of Aquatic Ecology, University of Girona, Campus de Montilivi, 17071, Girona, Spain

    L. Calvó, X. Vila, C. A. Abella & L. Jesús Garcia-Gil

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  1. L. Calvó
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  2. X. Vila
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  3. C. A. Abella
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  4. L. Jesús Garcia-Gil
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Correspondence to L. Jesús Garcia-Gil.

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Calvó, L., Vila, X., Abella, C.A. et al. Use of the ammonia-oxidizing bacterial-specific phylogenetic probe Nso1225 as a primer for fingerprint analysis of ammonia-oxidizer communities. Appl Microbiol Biotechnol 63, 715–721 (2004). https://doi.org/10.1007/s00253-003-1433-1

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  • DOI: https://doi.org/10.1007/s00253-003-1433-1

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