High-Throughput Isolation and Automated Sorting of Diverse Microbiomes Reduce Biases of Traditional Cultivation Strategies

Metagenomics has revolutionized our knowledge of the structure of gut-associated microbial communities and revealed interesting associations between health and disease. However, solely determining the taxonomic structure and functional potential through metagenomics is insufficient for understanding the mechanisms behind microbial community functioning. In order to obtain a mechanistic understanding of gut-associated health and disease, metagenomics must be supplemented with isolating pure strains from patient samples. However, traditional cultivation strategies are laborious; low-throughput; and feature biased sampling of taxa due to species being rare, slow-growing, or having specific metabolic requirements. To address these limitations, we developed a high-throughput anaerobic cultivation technology based on droplet microfluidics which stochastically encapsulates individual bacteria in droplets, cultivates the isolated organisms, and automatically sorts the droplets based on colony-density. Anaerobic droplet cultivation enabled several advantages over culture plates including: (1) an increase in the cultivated community richness by up to 410%, (2) a cultivated community representation which more closely resembles the input sample, (3) an enhancement in the number of strains cultivated which were <1% abundant in raw stool, and (4) cultivation of strains within the clinically important genus Bacteroides, which could not be grown on agar plates. Automated sorting of droplets for slow-growing colonies further enhanced the cultivated relative abundance of rare stool strains. In total, our method improves the cultivation of organisms broadly across phyla, including organisms which are rare and/or slow-growing, and can be used in conjunction with traditional microbiology techniques to increase the speed and efficiency of recovering species from complex microbial communities.