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Establishing anaerobic hydrocarbon-degrading enrichment cultures of microorganisms under strictly anoxic conditions

Nature Protocols volume 13, pages 1310–1330 (2018)Cite this article

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Abstract

Traditionally, the description of microorganisms starts with their isolation from an environmental sample. Many environmentally relevant anaerobic microorganisms grow very slowly, and often they rely on syntrophic interactions with other microorganisms. This impedes their isolation and characterization by classic microbiological techniques. We developed and applied an approach for the successive enrichment of syntrophic hydrocarbon-degrading microorganisms from environmental samples. We collected samples from microbial mat-covered hydrothermally heated hydrocarbon-rich sediments of the Guaymas Basin and mixed them with synthetic mineral medium to obtain sediment slurries. Supplementation with defined substrates (i.e., methane or butane), incubation at specific temperatures, and a regular maintenance procedure that included the measurement of metabolic products and stepwise dilutions enabled us to establish highly active, virtually sediment-free enrichment cultures of actively hydrocarbon-degrading communities in a 6-months to several-years' effort. Using methane as sole electron donor shifted the originally highly diverse microbial communities toward defined mixed cultures dominated by syntrophic consortia consisting of anaerobic methane-oxidizing archaea (ANME) and different sulfate-reducing bacteria. Cultivation with butane at 50 °C yielded consortia of archaea belonging to Candidatus Syntrophoarchaeum and Candidatus Desulfofervidus auxilii partner bacteria. This protocol also describes sampling for further molecular characterization of enrichment cultures by fluorescence in situ hybridization (FISH), and transcriptomics and metabolite analyses, which can provide insights into the functioning of hydrocarbon metabolism in archaea and resolve important mechanisms that enable electron transfer to their sulfate-reducing partner bacteria.

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Figure 1: Setups for the production of anaerobic medium (Step 1).
Figure 2
Figure 3: Setup for the temperature-controlled fixation of cell material for transcriptome analysis (Step 23C(ii–vi)).
Figure 4: The culture medium in serum bottles.
Figure 5: Appearance of enrichment cultures during cultivation.

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Acknowledgements

We thank F. Widdel for his advice and guidance in regard to cultivation and the development of this protocol. We are indebted to R. Appel, S. Menger and T. Holler for their contributions to the maintenance of the enrichment cultures and their suggestions for further improvement of our protocol. This protocol was tested with samples collected during Guaymas expedition AT15/45 in December 2016, Chief Scientist A. Teske, NSF grant BIO-OCE 1357238. This work was supported by the DFG Excellence Cluster MARUM and a Leibniz Grant to A. Boetius.

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Authors and Affiliations

  1. Max-Planck Institute for Marine Microbiology, Bremen, Germany

    Rafael Laso-Pérez, Viola Krukenberg & Gunter Wegener

  2. Alfred Wegener Institute, Helmholtz Centre for Polar and Marine Research, Bremerhaven, Germany

    Rafael Laso-Pérez, Viola Krukenberg & Gunter Wegener

  3. Helmholtz Centre for Environmental Research – UFZ, Leipzig, Germany

    Florin Musat

  4. MARUM, Center for Marine Environmental Sciences, University of Bremen, Bremen, Germany

    Gunter Wegener

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  1. Rafael Laso-Pérez
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  2. Viola Krukenberg
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  3. Florin Musat
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  4. Gunter Wegener
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Contributions

R.L.-P., V.K., F.M. and G.W. contributed equally to the design, validation and optimization of this protocol. R.L.-P., V.K. and G.W. prepared the figures; R.L.-P. and G.W. prepared the supplementary videos. R.L.-P., V.K., F.M. and G.W. cooperatively wrote and edited the manuscript.

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Correspondence to Gunter Wegener.

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The authors declare no competing financial interests.

Supplementary information

Step 1A.

Preparation of anoxic medium in a Widdel flask. (MOV 29836 kb)

Step 1B.

Preparation of anoxic medium in a Duran bottle. (MOV 29422 kb)

Step 8.

Addition of hydrocarbon substrates to culture bottles. (MOV 12646 kb)

Step 17B.

Enrichment culture transfer without an anoxic chamber. (MOV 20183 kb)

Step 23C.

Collection and preservation of material for RNA extraction using RNAlater. (MP4 25460 kb)

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Laso-Pérez, R., Krukenberg, V., Musat, F. et al. Establishing anaerobic hydrocarbon-degrading enrichment cultures of microorganisms under strictly anoxic conditions. Nat Protoc 13, 1310–1330 (2018). https://doi.org/10.1038/nprot.2018.030

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