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

BMC Microbiology

Methodology Article

Jan Mumme¹  Kathrin Heeg²  Edith Nettmann³  Michael Klocke⁴  Antje Fröhling⁵  Oliver Schlüter⁵ 

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

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

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

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

[5]Institute of Environmental Engineering, Ruhr University Bochum, Universitätsstrasse 150, 44780, Bochum, Germany

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

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

关键词: Flow cytometry;    in situ;    Flow-FISH;    Biogas reactor;    Upflow anaerobic solid state (UASS) reactor;    Anaerobic digestion;   

DOI  :  10.1186/1471-2180-13-278

 received in 2013-07-05, accepted in 2013-11-21,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundThe 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.ResultsSix 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.ConclusionsThe 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.

【 授权许可】

CC BY   
© Nettmann et al.; licensee BioMed Central Ltd. 2013