基于聚合物膜包封的微生物原位培养装置

An in-situ culture device for microorganisms based on polymer membrane encapsulation

摘要:

目的 传统培养方法仅能揭示微生物多样性的一小部分，大量微生物无法在实验室条件下培养。原位培养技术的出现为解决这一问题提供了关键手段，本研究旨在创新原位培养技术，研究未知培养条件下的微生物，并探究其在未培养微生物领域的潜在应用。方法 采用PCR管作为装置主体，引入“先分选，再培养”的概念，通过聚合物膜分隔微生物与环境进行独立培养。通过大肠杆菌纯培养实验验证了装置的有效性，并将其应用于土壤、污水和山泉水等不同环境的原位培养中。结果 验证实验结果显示，大肠杆菌纯培养装置内大肠杆菌的丰度显著增加。在聚合物膜浓度达到15%时，包封效果即可有效防止大肠杆菌逃逸，共培养实验进一步验证了这一结论。在原位培养实验中，成功应用所设计的装置从污水、土壤和山泉水样本中培养了单细胞微生物。测序结果表明，该方法可以培养在实验室中难以培养的物种。通过比对NCBI数据库，确认成功培养出了新的物种，证明了培养装置在不同环境下的有效性。结论 本研究的培养方法适用于单细胞微生物培养、特定微生物群落富集和多种微生物共培养。相比传统培养方法，该技术能分离并培养更多的微生物种类和数量。新技术不仅分离和培养出了更多微生物，还培养了之前无法在实验室条件下培养的微生物，对微生物学和生态学研究具有重要意义。

Abstract:

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.