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

Abstract

Our understanding of in situ microbial 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 underrepresented 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 slower-growing Negativicutes and Methanobacteria from 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.

Fig. 1. GrowMiDE: Double emulsion platform for bacterial cultivation.

A Schematic representation of the GrowMiDE platform. Four syringe pumps drive oil and aqueous solutions into a custom microfluidic device to encapsulate single microbes within 30 or 45 µm diameter double emulsions (DEs); DE generation is monitored in real-time by a high-speed camera attached to an Amscope stereoscope. B Schematic and representative brightfield image of DE droplets (scale bar = 30 µm). C Merged brightfield and fluorescent images of single E. coli-GFP cells loaded into DEs (left) and after growth in M9 + glucose for 24 h (right). D Brightfield microscopy images indicating growth of diverse anaerobes within DEs including the sulfate-reducer Desulfovibrio ferrophilus IS5 on 60 mM lactate and 30 mM NaSO₄, the acetogen Thermoanaerobacter kivui on 50 mM glucose at 65 °C, lactic acid-producing fermenter Lactococcus lactis NZ9000 on 50 mM glucose, and mixed acid fermenter E. coli MG1655 on 50 mM glucose.

Fig. 2. GrowMiDE enrichment of distinct microbial communities from human gut microbiota.

A Schematic overview of stool enrichments in DEs vs batch enrichments in mBHI. B Alpha diversity from input stool, GrowMiDE, and batch enrichments based on total unique ASVs. Floating bar plots represent the mean and range, n = 5–6. C Beta diversity from input stool, GrowMiDE, and batch enrichments based on type of unique ASVs. D Relative 16 S rRNA gene amplicon abundances from input stool samples, GrowMiDE enrichments, and batch enrichments from stool cell suspensions. E Relative 16 S rRNA gene amplicon abundances from a timecourse of stool enrichments in DE vs batch cultures sacrificed at 12, 24, 48, and 72 h, and corresponding absolute 16 S rRNA gene amplicon abundances of (F) M. smithii (class: Methanobacteria) and (G) P. faecium (class: Negativicutes). Detailed explanations of the enrichment conditions and samples types in Fig. 2D are outlined in Table S3.

Fig. 3. GrowMiDE can maintain and enrich growth yield specialists through nutrient privatization.

A Fermentation pathways of L. lactis WT (black) and ∆ldhA (red) strains in mixed batch cultures or GrowMiDE enrichments. B Growth of L. lactis monocultures and cocultures (1:1 starting ratio) on CDM + 25 mM glucose (n = 3, biological replicates, error bars indicate SEM). C Frequencies of populations across transfers in mixed batch cultures of L. lactis strains on 50 mM glucose. Each transfer received a 1% inoculum into fresh CDM + 50 mM glucose, and CFUs for each strain were determined from the grown community at each transfer (n = 3, biological replicates, error bars indicate SEM). D Cell densities across transfers in GrowMiDE enrichments of L. lactis strains on 50 mM glucose. Initial mixed culture contained ~80% ∆ldhA as determined by CFUs/mL on CDM + Ery⁵ plates. At the end of each transfer, the DEs were disrupted in bulk by incubating with 1H,1H,2H,2H-perfluoro-1-octanol (PFO), diluted to an OD = 0.05 to achieve single-cell loading, and cells were re-packaged into DEs for the next transfer. (n = 3, technical replicates, error bars indicate SEM). CFUs for each strain were determined from the grown community at each transfer to determine relative cell densities, and the initial mixed culture contained ~80% ∆ldhA prior to splitting into batch or DE competition experiments. All L. lactis transfers were incubated for 48 h at 30 °C.

Fig. 4. GrowMiDE + DE-FACS as a novel microbial enrichment and isolation platform.

A Overview and potential applications for microbial enrichments using GrowMiDE followed by DE-FACS to obtain isolates. B Relative ratios of 4 glucose-catabolizing strains in a mock community enriched in batch cultures or the GrowMiDE platform on CDM + 25 mM glucose for 48 h. n = 3. Error bars indicate SEM. C FACS profiles of 30 µm DEs containing the mock community encapsulated with 2.5 µM SYTO_bc after 48 h and subsequently sorted into individual wells on a 96-well plate. Plate wells were preloaded with growth medium and 10 uL of PFO to disrupt DEs. 767 events were gated within the top 2% of the SYTO⁺ DE population (38321 events) and 192 were sorted onto plates to assess for growth. D Growth curves of 34 representative output isolates downstream of DE-FACS. All growth assays were performed at 30 °C.