High-throughput Single-cell Cultivation Reveals Underexplored Rare Biosphere in Deep-sea Sediments Along the Southwest Indian Ridge

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 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 the isolation efficiency with a semi-automated droplet picker, and improved humidity control to enable long-term cultivation with an oligotrophic medium for up to five months according to the slow-growing nature of most deep-sea species. The improved MSP method allows isolation of microbes by picking and investigation of microbial diversity by high throughput sequencing the pooled sample cultures. By picking of 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 at SWIR, including 15 potential novel species. On the other hand, based on 16S rRNA amplicon sequencing analysis, the microbial diversity of MSP-pooled samples account for 20.5% of the total OTUs detected by sequencing of original and cultivated samples. Moreover, the MSP-pooled method revealed 639 OTUs belonging to the rare biosphere with a relative abundance of < 0.01% in the original samples, including 384 OTUs that were absent from the original sample sequence, which accounted for 18.7% of the total OTUs. In comparison, agar plate cultivation only recovered 8.69% of the total microbial diversity and 245 OTUs with a relative abundance of < 0.01%. Our results indicate the high robustness and efficiency of the improved MSP platform at revealing the environmentally rare biosphere, especially for slow-growing species. Overall, the MSP platform had 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.