Microorganisms in the deep sea play vital roles in marine ecosystems. However, despite great advances brought by high throughput sequencing and metagenomics, only a small portion of microorganisms living in the environment can be cultivated in the laboratory and systematically studied. In this study, an improved high-throughput microfluidic streak plate (MSP) platform was developed to speed up the isolation of microorganisms from deep-sea sediments and evaluated with deep-sea sediments collected from the Southwest Indian Ridge (SWIR). Based on our previously reported MSP method, we improved its isolation efficiency with a semi-automated droplet picker and improved humidity control to enable long-term cultivation with a low-nutrient medium for up to five months according to the slow-growing nature of most deep-sea species. The improved MSP method allows the isolation of microbes by selection and investigation of microbial diversity by high throughput sequencing of the pooled sample cultures. By picking individual droplets and scale-up cultivation, a total of 772 strains that were taxonomically assigned to 70 species were isolated from the deep-sea sediments in the SWIR, including 15 potential novel species. On the other hand, based on 16S rRNA gene amplicon sequencing analysis, the microbial diversity of the SWIR was studied and documented with culture-dependent and independent methods in this study. The superiority of the MSP platform in revealing the rare biosphere was also evaluated based on amplicon sequencing. The results show that droplet-based single-cell cultivation of the MSP has a much higher ability than traditional agar plate cultivation in obtaining microbial species and more than 90% of operational taxonomic units (OTUs) detected in the MSP pool belong to the rare biosphere. Our results indicate the high robustness and efficiency of the improved MSP platform in revealing the environmentally rare biosphere, especially for slow-growing species. Overall, the MSP platform has a superior ability to recover microbial diversity than conventional agar plates and it was found to hold great potential for recovering rare microbial resources from various environments.

中文翻译：

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高通量单细胞培养揭示了西南印第安海岭沿岸深海沉积物中未开发的稀有生物圈。

深海中的微生物在海洋生态系统中起着至关重要的作用。但是，尽管高通量测序和宏基因组学带来了长足的进步，但只能在实验室中培养和系统地研究生活在环境中的一小部分微生物。在这项研究中，开发了一种改进的高通量微流控条纹板（MSP）平台，以加快从深海沉积物中分离微生物的速度，并利用从西南印第安海岭（SWIR）收集的深海沉积物进行评估。根据我们先前报道的MSP方法，我们根据半自动液滴拾取器的半自动生长特性，通过半自动液滴拾取器提高了分离效率，并改善了湿度控制，从而可以在低营养的培养基下进行长达五个月的长期培养。深海物种。改进的MSP方法可以通过合并样品培养物的高通量测序，通过选择和研究微生物多样性来分离微生物。通过挑选单个飞沫并扩大规模，从SWIR的深海沉积物中分离出了共分类为70种的772个菌株，其中包括15种潜在的新物种。另一方面，基于16S rRNA基因扩增子测序分析，研究了SWIR的微生物多样性，并采用了依赖于培养物的方法进行了记录。还基于扩增子测序评估了MSP平台在揭示稀有生物圈方面的优越性。结果表明，基于液滴的单细胞培养MSP具有比传统琼脂平板培养更高的获得微生物种类的能力，并且MSP池中检测到的90％以上的操作生物分类单位（OTU）属于稀有生物圈。 。我们的结果表明，改进的MSP平台具有很高的鲁棒性和效率，可揭示环境稀有的生物圈，特别是对于生长缓慢的物种。总体而言，MSP平台具有比传统琼脂平板更高的恢复微生物多样性的能力，并且发现它具有从各种环境中恢复稀有微生物资源的巨大潜力。我们的结果表明，改进的MSP平台具有很高的鲁棒性和效率，可揭示环境稀有的生物圈，特别是对于生长缓慢的物种。总体而言，MSP平台具有比传统琼脂平板更高的恢复微生物多样性的能力，并且发现它具有从各种环境中恢复稀有微生物资源的巨大潜力。我们的结果表明，改进的MSP平台具有很高的鲁棒性和效率，可揭示环境稀有的生物圈，特别是对于生长缓慢的物种。总体而言，MSP平台具有比传统琼脂平板更高的恢复微生物多样性的能力，并且发现它具有从各种环境中恢复稀有微生物资源的巨大潜力。