Abstract
The isolation chip method (iChip) provides a novel approach for culturing previously uncultivable microorganisms; this method is currently limited by the user being unable to ensure single-cell loading within individual wells. To address this limitation, we integrated flow cytometry-based fluorescence-activated cell sorting with a modified iChip (FACS-iChip) to effectively mine microbial dark matter in soils. This method was used for paddy soils with the aim of mining uncultivable microorganisms and making preliminary comparisons between the cultured microorganisms and the bulk soil via 16S rRNA gene sequencing. Results showed that the FACS-iChip achieved a culture recovery rate of almost 40% and a culture retrieval rate of 25%. Although nearly 500 strains were cultured from 19 genera with 8 FACS-iChip plates, only six genera could be identified via 16S rRNA gene amplification. This result suggests that the FACS-iChip is capable of detecting strains in the currently dead spaces of PCR-based sequencing technology. We, therefore, conclude that the FACS-iChip system provides a highly efficient and readily available approach for microbial ‘dark matter’ mining.
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11 November 2020
A Correction to this paper has been published: https://doi.org/10.1007/s42995-020-00079-3
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Acknowledgements
This research was financially supported by the National Natural Science Foundation of China (41991334), the Zhejiang Provincial Natural Science Foundation of China (LD19D060001, LQ20C030006) and the China Postdoctoral Science Foundation (2019M652097).
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BM, RX and JX designed this study; HL and YW performed the experiments and HL wrote the manuscipt; ES revised the manuscript; SY drew the figure 1; all authors read and approved the final manuscript.
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Edited by Chengchao Chen.
The original online version of this article was revised: The presentation of Table 2 was incorrect.
SPECIAL TOPIC: Cultivation of the uncultured microorganisms.
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Liu, H., Xue, R., Wang, Y. et al. FACS-iChip: a high-efficiency iChip system for microbial ‘dark matter’ mining. Mar Life Sci Technol 3, 162–168 (2021). https://doi.org/10.1007/s42995-020-00067-7
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DOI: https://doi.org/10.1007/s42995-020-00067-7