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Development and application of real-time PCR for quantification of specific ammonia-oxidizing bacteria in activated sludge of sewage treatment systems

  • Applied Genetics and Molecular Biotechnology
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Abstract

In this study, four real-time polymerase chain reaction (PCR) primer sets were developed for the 16S rRNA genes of specific ammonia-oxidizing bacteria (AOB) found in activated sludge of sewage treatment systems. The primer sets target two of several sequence types of the Nitrosomonas oligotropha cluster, members within the Nitrosomonas communis cluster, and all members of the Nitrosomonas europaea–Nitrosococcus mobilis cluster. The detection limit of each primer set was in the range of 3×101–6×102 genes reaction−1. Reliable quantification of the target AOB DNA was obtained when the target AOB DNA comprised more than 0.1% of total AOB DNA in the sample. The application of the primer sets to samples taken from five sewage treatment systems showed that, in all systems, the majority of the AOB population was comprised of one sequence type of the N. oligotropha cluster (3.9±1.5×109–1.7±0.5×1010 cell l−1) and, in most systems, followed by members within the N. communis cluster (2.8±0.3×109–1.0±0.1×1010 cell l−1) or/and another sequence type of the N. oligotropha cluster (1.5±0.6×108–5.5±0.5×108 cell l−1). N. europaea–N. mobilis cluster arose solely in small numbers (4.9±0.8×108 cell l−1) in one system. Real-time PCR-amplified products obtained from genomic DNA extracted from samples were verified using clone library, and it revealed that only the target AOB DNA were PCR amplified, without amplification of the nontarget sequences.

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Acknowledgements

We are grateful to the Tokyo Metropolitan Government for providing samples and data from the sewage treatment systems. We would also like to extend special thanks to Yoriko Sakamoto for providing laboratory assistance.

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Authors and Affiliations

  1. Department of Urban Engineering, Graduate School of Engineering, the University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan

    Tawan Limpiyakorn

  2. Research Center for Water Environment Technology, Graduate School of Engineering, the University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan

    Futoshi Kurisu & Osami Yagi

  3. National Research Center for Environmental and Hazardous Waste Management, Institute Building 2, Chulalongkorn University, Phayathai Road, Pathumwan, Bangkok, 10330, Thailand

    Tawan Limpiyakorn

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  1. Tawan Limpiyakorn
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  2. Futoshi Kurisu
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  3. Osami Yagi
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Correspondence to Tawan Limpiyakorn.

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Limpiyakorn, T., Kurisu, F. & Yagi, O. Development and application of real-time PCR for quantification of specific ammonia-oxidizing bacteria in activated sludge of sewage treatment systems. Appl Microbiol Biotechnol 72, 1004–1013 (2006). https://doi.org/10.1007/s00253-006-0366-x

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  • DOI: https://doi.org/10.1007/s00253-006-0366-x

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