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Cultivation of previously uncultured microorganisms with a continuous-flow down-flow hanging sponge (DHS) bioreactor, using a syntrophic archaeon culture obtained from deep marine sediment as a case study

Nature Protocols volume 17, pages 2784–2814 (2022)Cite this article

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Abstract

In microbiology, cultivation is a central approach for uncovering novel physiology, ecology, and evolution of microorganisms, but conventional methods have left many microorganisms found in nature uncultured. To overcome the limitations of traditional methods and culture indigenous microorganisms, we applied a two-stage approach: enrichment/activation of indigenous organisms by using a continuous-flow down-flow hanging sponge bioreactor and subsequent selective batch cultivation. Here, we provide a protocol for this bioreactor-mediated technique using activation of deep marine sediment microorganisms and downstream isolation of a syntrophic co-culture containing an archaeon closely related to the eukaryote ancestor (Candidatus Promethearchaeum syntrophicum strain MK-D1) as an example. Both stages can easily be tailored to target other environments and organisms by modifying the inoculum, feed solution/gases, attachment material and/or cultivation media. We anaerobically incubate polyurethane sponges inoculated with deep-sea methane seep sediment in a reactor at 10 °C and feed anaerobic artificial seawater medium and methane. Once phylogenetically diverse and metabolically active microorganisms are adapted to synthetic conditions in the reactor, we transition to growing community samples in glass tubes with the above medium, simple substrates and selective compounds (e.g., antibiotics). To accommodate for the slow growth anticipated for target organisms, primary cultures can be incubated for ≥6–12 months and analyzed for community composition even when no cell turbidity is observed. One casamino acid- and antibiotic-amended culture prepared in this way led to the enrichment of uncultured archaea. Through successive transfer in vitro combined with molecular growth monitoring, we successfully obtained the target archaeon with its partner methanogen as a pure syntrophic co-culture.

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Fig. 1: The DHS bioreactor.
Fig. 2
Fig. 3: Overview of the DHS bioreactor system.
Fig. 4: The culture medium in serum vials.
Fig. 5: Growth curves of strain MK-D1 in anaerobic medium supplemented with casamino acids (0.05% (wt/vol)) and powdered milk (0.1% (wt/vol)).

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Data availability

The main data discussed in this protocol are available in the supporting primary research papers25,30.

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Acknowledgements

We thank H. Harada, H. Sumino, M. Takahashi, Y. Takahashi, M. Hatamoto and T. Yamaguchi for their advice and guidance in the development of the protocol. We also thank A. Miyashita, Y. Yashiro, K. Aoi, M. Ehara, M. Aoki, Yayoi Saito, N. Nakahara, M. Isozaki, S. A. Connon, V. J. Orphan, J. Amend and L. M. Momper for their contribution to the maintenance of the DHS reactor and their suggestions for further improvement of the protocol and Y. Tsuchiya for his help with movie editing. This protocol was tested with a sample collected during the cruise YK06-03 (JAMSTEC) in May 2006. This work was partially supported by grants from the Japan Society for the Promotion of Science (JSPS) (KAKENHI Grants JP19H01005 to H.I., JP18H03367 to M.K.N. and JP22H04985 to H.I. and M.K.N.) and the Moore–Simon Project on the Origin of the Eukaryotic Cell (GBMF9743 to H.I. and M.K.N.).

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  1. These authors contributed equally: Hiroyuki Imachi, Masaru K. Nobu, Masayuki Miyazaki.

Authors and Affiliations

  1. Institute for Extra-cutting-edge Science and Technology Avant-garde Research (X-star), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Japan

    Hiroyuki Imachi, Masayuki Miyazaki, Eiji Tasumi, Yumi Saito, Sanae Sakai, Miyuki Ogawara & Ken Takai

  2. Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan

    Masaru K. Nobu

  3. Department of Civil and Environmental Engineering, Graduate School of Advanced Science and Engineering, Hiroshima University, Higashi-Hiroshima, Japan

    Akiyoshi Ohashi

  4. Section for Exploration of Life in Extreme Environments, Exploratory Research Center on Life and Living Systems (ExCELLS), National Institute of Natural Sciences, Okazaki, Japan

    Ken Takai

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Contributions

H.I., M.K.N. and M.M. contributed equally to the design, validation and optimization of the protocol. H.I., M.K.N., M.M, Y.S. and A.O. prepared the tables and figures; H.I., M.M., E.T., S.S., Y.S., and M.O. prepared the supplementary videos. H.I., M.K.N., M.M. and K.T. wrote the manuscript. All authors read and approved the manuscript submission.

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Correspondence to Hiroyuki Imachi, Masaru K. Nobu or Masayuki Miyazaki.

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Key references using this protocol

Imachi, H. et al. Nature 577, 519–525 (2020): https://doi.org/10.1038/s41586-019-1916-6

Aoki, M. et al. PLoS ONE 9, e105356 (2014): https://doi.org/10.1371/journal.pone.0105356

Imachi, H. et al. ISME J. 5, 1913–1925 (2011): https://doi.org/10.1038/ismej.2011.64

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Imachi, H., Nobu, M.K., Miyazaki, M. et al. Cultivation of previously uncultured microorganisms with a continuous-flow down-flow hanging sponge (DHS) bioreactor, using a syntrophic archaeon culture obtained from deep marine sediment as a case study. Nat Protoc 17, 2784–2814 (2022). https://doi.org/10.1038/s41596-022-00735-1

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