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[Objective] Conventional culture methods can merely uncover a fraction of microbial diversity. A vast number of microorganisms remain unculturable under laboratory settings. The advent of in-situ culture technology offers a key solution to this predicament. This study endeavors to innovate the in-situ culture technology, explore microorganisms under hitherto unknown culture conditions, and probe into their potential applications within the realm of uncultured microorganisms. [Methods] PCR tubes were employed as the core of the device, and the idea of sorting prior to culture was introduced. Microorganisms were separated from the environment by means of polymer membranes for independent culture. The efficacy of the device was validated through the pure culture of Escherichia coli. Moreover, this device was applied to in-situ culture in diverse environments such as soil, sewage, and mountain spring water. [Results] The abundance of E. coli increased significantly in the pure-culture device. When the concentration of the polymer membrane reached 15%, the encapsulation effect effectively prevented the escape of E. coli. The co-culture experiments further corroborated this finding. In the in-situ culture experiments, the device designed in this study successfully cultured single-cell microorganisms from sewage, soil, and mountain spring water samples. Sequencing results indicated that this device could culture species recalcitrant to be cultured in the laboratory. Comparison with the NCBI database verified that new species were successfully cultured, which demonstrated the effectiveness of the culture device in various environments. [Conclusion] The culture method designed in this study is suitable for single-cell microbial culture, enrichment of specific microbial communities, and co-culture of multiple microorganisms. The device can isolate and culture richer and more microorganisms than conventional culture methods. This new technology not only isolates and cultures more microorganisms but also manages to culture those previously unculturable under laboratory conditions. It holds great significance for microbiological and ecological research.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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