Applied Microbiology and Biotechnology

, Volume 102, Issue 13, pp 5707–5715 | Cite as

Rapid isolation of fast-growing methanotrophs from environmental samples using continuous cultivation with gradually increased dilution rates

  • Jisun Kim
  • DaeHyun D. Kim
  • Sukhwan Yoon
Methods and protocols


Methanotrophs have recently gained interest as biocatalysts for mitigation of greenhouse gas emission and conversion of methane to value-added products; however, their slow growth has, at least partially, hindered their industrial application. A rapid isolation technique that specifically screens for the fastest-growing methanotrophs was developed using continuous cultivation with gradually increased dilution rates. Environmental samples collected from methane-rich environments were enriched in continuously stirred tank reactors with unrestricted supply of methane and air. The reactor was started at the dilution rate of 0.1 h−1, and the dilution rates were increased with an increment of 0.05 h−1 until the reactor was completely washed out. The shifts in the overall microbial population and methanotrophic community at each step of the isolation procedure were monitored with 16S rRNA amplicon sequencing. The predominant methanotrophic groups recovered after reactor operations were affiliated to the gammaproteobacterial genera Methylomonas and Methylosarcina. The methanotrophic strains isolated from the reactor samples collected at their respective highest dilution rates exhibited specific growth rates up to 0.40 h−1; the highest value reported for methanotrophs. The novel isolation method developed in this study significantly shortened the time and efforts needed for isolation of methanotrophs from environmental samples and was capable of screening for the methanotrophs with the fastest growth rates.


Methane Methanotrophs Continuous cultivation Growth rate Isolation 



This research was financially supported by the National Research Foundation of Korea (NRF) (grant no. 2015M3D3A1A01064881) and the “R&D Center for Reduction of Non-CO2 Greenhouse Gases” (grant no. 2017002420002) funded by Korea Ministry of Environment (MOE) as “Global Top Environment R&D Program”. The authors were also financially supported by Korea Ministry of Land, Infrastructure and Transport (MOLIT) as U-City Master and Doctor Course Grant Program and the Brain Korea 21 Plus Project (grant no. 21A20132000003).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_8978_MOESM1_ESM.xlsx (135 kb)
ESM 1 (XLSX 134 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Civil and Environmental EngineeringDaejeonSouth Korea

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