Abstract
Methanogenic archaea play a crucial role in the global carbon cycle and in climate change. Recent metagenomic sequencing has revealed a considerable number of (putative) H2-dependent methylotrophic methanogens (HMMs) across the archaeal tree and in diverse environments. Traditional isolation methods, such as dilution-to-extinction and roll-tube techniques, fail to cultivate fastidious HMMs. Here, we describe a four-stage isolation strategy designed to selectively isolate HMMs by using a flexible combination of methods to systematically reduce microbial complexity to a pure culture. In the initial stage, the growth conditions for the target HMM were optimized through closed-batch cultivation encompassing >50 conditions. Second, HMM-containing cultures were serially diluted in 96-well plates combined with substrate limitation to eliminate non-target archaea. In stage 3, the bacterial diversity in the culture was further decreased to a single bacterium by treatment with antibiotics and lysozyme. Finally, a last bacterial contaminant was removed by repeated addition of antibiotic mixtures and successive dilution transfers, leading to the successful isolation of the first pure culture of Methanosuratincola petrocarbonis LWZ-6, an HMM of the phylum Thermoproteota. This protocol also describes molecular methods, including 16S rRNA gene amplicon sequencing, metagenome sequencing and quantitative PCR, to track microbial community shifts and assess the growth advantage of the target HMM, enabling monitoring of the stepwise elimination of non-target microorganisms and ultimately confirming the purification of the target HMM. The duration of the protocol will vary for different HMMs depending on their substrate utilization, growth rate and method selection.
Key points
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This protocol details a four-stage strategy to selectively isolate H2-dependent methylotrophic methanogens by creating a relative growth advantage of target microorganism over different non-target microbial groups in each stage to eliminate non-target microorganisms.
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This protocol advances traditional isolation methods to enable the culture of fastidious HMMs.
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Data availability
The main data discussed in this protocol are available in the supporting primary research paper31.
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Acknowledgements
We thank Jie Wang for the supplementary video preparation. This study was supported by the National Natural Science Foundation of China (32325002 and 92351301), the Agricultural Science and Technology Innovation Project of the Chinese Academy of Agriculture Science (CAAS-ASTIP-2021-BIOMA-01, CAAS-CSGLCA-202301 and CAAS-ZDRW202305), the Central Public-interest Scientific Institution Basal Research Fund (Y2024PTO3) and the Sichuan Province Science and Technology Department (2024NSFTD0020).
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L.C. initiated this study, and L.C., K.W. and L.Z. designed the research. L.L. performed the initial cultivation. K.W., L.Z., L.L., J.L. and M.Y. performed the isolation process. L.C., K.W. and L.Z. designed the figures. L.Z. and S.M. prepared the supplementary video. K.W., L.Z., L.C. and D.Z.S wrote the manuscript with contributions from all the authors.
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Wu, K. et al. Nature 632, 1124–1130 (2024): https://doi.org/10.1038/s41586-024-07728-y
Supplementary information
Supplementary Video 1
Preparation of sterile anoxic stock solutions
Supplementary Video 2
Preparation of pre-reduced medium in serum bottles
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Wu, K., Zhou, L., Liu, L. et al. Targeted isolation of H2-dependent methylotrophic methanogens by a cocktail approach. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01224-x
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DOI: https://doi.org/10.1038/s41596-025-01224-x
