Double emulsions as a high-throughput enrichment and isolation platform for slower-growing microbes

Abstract

Our understanding ofin situmicrobial physiology is primarily based on physiological characterization of fast-growing and readily-isolatable microbes. Microbial enrichments to obtain novel isolates with slower growth rates or physiologies adapted to low nutrient environments are plagued by intrinsic biases for fastest-growing species when using standard laboratory isolation protocols. New cultivation tools to minimize these biases and enrich for less well-studied taxa are needed. In this study, we developed a high-throughput bacterial enrichment platform based on single cell encapsulation and growth within double emulsions (GrowMiDE). We showed that GrowMiDE can cultivate many different microorganisms and enrich for novel taxa that are never observed in traditional batch enrichments. For example, preventing dominance of the enrichment by fast-growing microbes due to nutrient privatization within the double emulsion droplets allowed cultivation of novelNegativicutesandMethanobacteriafrom stool samples in rich media enrichment cultures. In competition experiments between growth rate and growth yield specialist strains, GrowMiDE enrichments prevented competition for shared nutrient pools and enriched for slower-growing but more efficient strains. Finally, we demonstrated the compatibility of GrowMiDE with commercial fluorescence-activated cell sorting (FACS) to obtain isolates from GrowMiDE enrichments. Together, GrowMiDE + DE-FACS is a promising new high-throughput enrichment platform that can be easily applied to diverse microbial enrichments or screens.

Advances in Culturomics Research on the Human Gut Microbiome: Optimizing Medium Composition and Culture Techniques for Enhanced Microbial Discovery

“…This methodology allows the cultivation of a diverse array of microbes, including taxa that are unrepresented in traditional batch cultures. For instance, with the prevention of nutrient monopolization by fast-growing microbes in double emulsion droplets, it is possible to cultivate slow-growing Negativicutes and Methanobacteria in enriched media cultures [51]. These innovative technological strides continue to redefine the boundaries of microbial cultivation, fostering enhanced diversity and a deeper understanding of microbial ecology.…”

Drop-by-drop Addition of Reagents to a Double Emulsion

“…Material transport across the oil shell has been applied for small molecule diffusion following an in-drop reaction to interface incompatible steps or for nutrient exchange to enable long-term cultivation of microbes. 32,33 Further, DEs are robust against coalescence. 20 In response to stresses, SE compartments merge with each other and combine, which results in reduced monodispersity and a loss of single entity resolution.…”

“…Consequently, DEs have been shown to be compatible with broadly accessible flow cytometry instruments. [21][22][23][24][25][26] When compared with equivalent SE droplet screening platforms, DE-flow cytometry offers a substantial improvement in terms of availability, functionality, and ease of use. SE droplet screening requires a custom-built sorting stand and significant microfluidic experience to achieve 1-3 color fluorescence detection and ~200 Hz sorting speeds.…”