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Molecular Analysis of the Microbial Community Developing in Continuous Culture of Methylococcus sp. Concept-8 on Natural Gas

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Abstract—

The composition of the microbial community formed in the course of long-term (over 60 days) continuous cultivation of an obligate methanotroph Methylococcus sp. Concept-8 on natural gas in a 50-L bioreactor at 42°C and pH 5.6 was monitored. Stable growth with high optical density of the culture (average OD540 = 13.5), specific growth rate of 0.2 h–1, and biomass yield of 4.0–4.5 g dry matter L–1 continued for 50 days. During the subsequent period of regime instability, optical density decreased sharply, down to OD540 = 4. Formate, acetate, and a number of unidentified fatty acids derived from oxidation of methane homologs were detected in the culture liquid. Microscopy revealed the highest share of satellite bacteria (10–15% of the total cell number) during the period of stable growth, while it dropped to 4.5% during growth decline. Molecular profiling of the community composition based on 16S rRNA gene sequence analysis revealed members of the genera Cohnella, Brevibacillus, Azospirillim, Thermomonas, Cupriavidus, and Paenibacillus among the major microbial satellites responsible for utilization of metabolites and the products of lysis of the methanotroph cells. During the phase of growth instability, the relative abundance of Brevibacillus decreased drastically; these organisms were most likely responsible for acetate oxidation. Isolates obtained by means of cultivation represented only a minor part of microbial diversity revealed in the community by molecular analysis. Isolation of the cultures of Brevibacillus and Cupriavidus species representing the functionally important components of the community opens the way for targeted construction of associations with strain Concept-8, which will display high productivity and stable growth.

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ACKNOWLEDGMENTS

The authors are grateful to V.V. Kevbrin for his assistance in identification of organic compounds in the culture liquid.

Funding

The work was supported by the Ministry of Education and Science of the Russian Federation, Federal Target Program grant no. 075-15-2019-1830, unique project ID RFMEFI60719X0297. Electron microscopy was performed at the Center for Collective Use of the Collection of Microorganisms UNIQEM, Federal Research Center for Biotechnology of the Russian Academy of Sciences. Molecular studies were carried out at the Collective Use Center “Bioengineering”, Federal Research Center for Biotechnology of the Russian Academy of Sciences.

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Authors and Affiliations

  1. Winogradsky Institute of Microbiology, Research Center of Biotechnology, Russian Academy of Sciences, 119071, Moscow, Russia

    I. Yu. Oshkin, S. E. Belova, E. N. Tikhonova, N. V. Pimenov & S. N. Dedysh

  2. Gazprom VNIIGAZ, 142717, Razvilka, Moscow oblast, Russia

    N. S. Khokhlachev, V. A. Semenova & O. P. Chervyakova

  3. NPO Biosintez, 127018, Moscow, Russia

    D. V. Chernushkin

  4. Institute of Bioengineering, Research Center of Biotechnology, Russian Academy of Sciences, 119071, Moscow, Russia

    A. V. Mardanov & N. V. Ravin

  5. Bach Institute of Biochemistry, Research Center of Biotechnology, Russian Academy of Sciences, 119071, Moscow, Russia

    V. O. Popov

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  1. I. Yu. Oshkin
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  2. S. E. Belova
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  3. N. S. Khokhlachev
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  7. E. N. Tikhonova
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  8. A. V. Mardanov
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  9. N. V. Ravin
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  10. V. O. Popov
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  11. N. V. Pimenov
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  12. S. N. Dedysh
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Corresponding author

Correspondence to S. N. Dedysh.

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Conflict of interests. The authors declare that they have no conflict of interest.

The authors’ contribution. N.S. Khokhlachev, V.A. Se-menova, O.P. Chervyakova, and D.V. Chernushkin conducted continuous cultivation and growth monitoring of strain Concept-8. Sample collection and data analysis were performed by I.Yu. Oshkin. S.E. Belova was in charge of cell counts by microscopy and isolation of satellite bacteria. Electron microscopy was performed by E.N. Tikhonova. A.V. Mardanov and N.V. Ravin were in charge of DNA isolation, sequencing, and primary data analysis. The text of the manuscript was written by S.N. Dedysh, I.Yu. Oshkin, V.O. Popov, and N.V. Pimenov. All authors participated in the discussion of the results.

Statement on the welfare of animals. This article does not contain any studies involving animals or human participants performed by any of the authors.

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Translated by E. Makeeva

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Oshkin, I.Y., Belova, S.E., Khokhlachev, N.S. et al. Molecular Analysis of the Microbial Community Developing in Continuous Culture of Methylococcus sp. Concept-8 on Natural Gas. Microbiology 89, 551–559 (2020). https://doi.org/10.1134/S0026261720050173

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