Open Access Methodology article

Development of a flow-fluorescence in situ hybridization protocol for the analysis of microbial communities in anaerobic fermentation liquor

Edith Nettmann12*, Antje Fröhling3, Kathrin Heeg14, Michael Klocke5, Oliver Schlüter3 and Jan Mumme1

Author Affiliations

1 APECS junior research group, Leibniz Institute for Agricultural Engineering, Max-Eyth-Allee 100, 14469 Potsdam, Germany

2 Institute of Environmental Engineering, Ruhr University Bochum, Universitätsstrasse 150, 44780 Bochum, Germany

3 Quality and Safety of Food and Feed, Leibniz Institute for Agricultural Engineering, Max-Eyth-Allee 100, 14469 Potsdam, Germany

4 Department Bioengineering, Leibniz Institute for Agricultural Engineering, Max-Eyth-Allee 100, 14469 Potsdam, Germany

5 Faculty of Process Sciences, Institute of Technical Environmental Protection, Environmental Microbiology, Technical University Berlin, Ernst-Reuter-Platz 1, 10587 Berlin, Germany

For all author emails, please log on.

BMC Microbiology 2013, 13:278  doi:10.1186/1471-2180-13-278

Published: 4 December 2013



The production of bio-methane from renewable raw material is of high interest because of the increasing scarcity of fossil fuels. The process of biomethanation is based on the inter- and intraspecific metabolic activity of a highly diverse and dynamic microbial community. The community structure of the microbial biocenosis varies between different biogas reactors and the knowledge about these microbial communities is still fragmentary. However, up to now no approaches are available allowing a fast and reliable access to the microbial community structure. Hence, the aim of this study was to originate a Flow-FISH protocol, namely a combination of flow cytometry and fluorescence in situ hybridization, for the analysis of the metabolically active microorganisms in biogas reactor samples. With respect to the heterogenic texture of biogas reactor samples and to collect all cells including those of cell aggregates and biofilms the development of a preceding purification procedure was indispensable.


Six different purification procedures with in total 29 modifications were tested. The optimized purification procedure combines the use of the detergent sodium hexametaphosphate with ultrasonic treatment and a final filtration step. By this treatment, the detachment of microbial cells from particles as well as the disbandment of cell aggregates was obtained at minimized cell loss. A Flow-FISH protocol was developed avoiding dehydration and minimizing centrifugation steps. In the exemplary application of this protocol on pure cultures as well as biogas reactor samples high hybridization rates were achieved for commonly established domain specific oligonucleotide probes enabling the specific detection of metabolically active bacteria and archaea. Cross hybridization and autofluorescence effects could be excluded by the use of a nonsense probe and negative controls, respectively.


The approach described in this study enables for the first time the analysis of the metabolically active fraction of the microbial communities within biogas reactors by Flow-FISH.

Flow cytometry; Fluorescence in situ hybridization; Flow-FISH; Biogas reactor; Upflow anaerobic solid state (UASS) reactor; Anaerobic digestion