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Design and application of a synthetic DNA standard for real-time PCR analysis of microbial communities in a biogas digester

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Abstract

A synthetic DNA fragment containing primer binding sites for the quantification of ten different microbial groups was constructed and evaluated as a reliable enumeration standard for quantitative real-time PCR (qPCR) analyses. This approach has been exemplary verified for the quantification of several methanogenic orders and families in a series of samples drawn from a mesophilic biogas plant. Furthermore, the total amounts of bacteria as well as the number of sulfate-reducing and propionic acid bacteria as potential methanogenic interaction partners were successfully determined. The obtained results indicated a highly dynamic microbial community structure which was distinctly affected by the organic loading rate, the substrate selection, and the amount of free volatile fatty acids in the fermenter. Methanosarcinales was the most predominant methanogenic order during the 3 months of observation despite fluctuating process conditions. During all trials, the modified quantification standard indicated a maximum of reproducibility and efficiency, enabling this method to open up a wide range of novel application options.

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Acknowledgments

We would like to thank the Deutsches Biomasseforschungszentrum gemeinnützige GmbH (DBFZ) for supplying and operating the experimental reactor and the provided laboratory analyses. This work was financially supported by the Fachagentur Nachwachsende Rohstoffe e.V. (FKZ 22001913), Germany.

Compliance with ethical standards

This article does not contain any studies with human participants or animals performed by any of the authors.

Funding

This study was funded by Fachagentur Nachwachsende Rohstoffe e.V., Germany (grant number FKZ 22001913).

Conflict of interest

The authors declare that they have no competing interests.

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Authors and Affiliations

  1. Institute of Microbiology and Wine Research, Johannes Gutenberg University Mainz, Johann-Joachim Becherweg 15, 55128, Mainz, Germany

    T. May, M. Koch-Singenstreu, J. Ebling, R. Stantscheff & H. König

  2. Deutsches Biomasseforschungszentrum gemeinnützige GmbH (DBFZ), Torgauer Straße 116, 04347, Leipzig, Germany

    L. Müller & F. Jacobi

  3. Institute of Earthsciences, Ruprecht-Karls University Heidelberg, Im Neuenheimer Feld 234-236, 69120, Heidelberg, Germany

    D. Polag & F. Keppler

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  1. T. May
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  2. M. Koch-Singenstreu
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  3. J. Ebling
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  4. R. Stantscheff
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  5. L. Müller
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  7. D. Polag
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  8. F. Keppler
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  9. H. König
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Correspondence to T. May.

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May, T., Koch-Singenstreu, M., Ebling, J. et al. Design and application of a synthetic DNA standard for real-time PCR analysis of microbial communities in a biogas digester. Appl Microbiol Biotechnol 99, 6855–6863 (2015). https://doi.org/10.1007/s00253-015-6721-z

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  • DOI: https://doi.org/10.1007/s00253-015-6721-z

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