Microdroplet-enabled highly parallel co-cultivation of microbial communities

Abstract

Microbial interactions in natural microbiota are, in many cases, crucial for the sustenance of the communities, but the precise nature of these interactions remain largely unknown because of the inherent complexity and difficulties in laboratory cultivation. Conventional pure culture-oriented cultivation does not account for these interactions mediated by small molecules, which severely limits its utility in cultivating and studying "unculturable" microorganisms from synergistic communities. In this study, we developed a simple microfluidic device for highly parallel co-cultivation of symbiotic microbial communities and demonstrated its effectiveness in discovering synergistic interactions among microbes. Using aqueous micro-droplets dispersed in a continuous oil phase, the device could readily encapsulate and co-cultivate subsets of a community. A large number of droplets, up to ∼1,400 in a 10 mm × 5 mm chamber, were generated with a frequency of 500 droplets/sec. A synthetic model system consisting of cross-feeding E. coli mutants was used to mimic compositions of symbionts and other microbes in natural microbial communities. Our device was able to detect a pair-wise symbiotic relationship when one partner accounted for as low as 1% of the total population or each symbiont was about 3% of the artificial community.

Figure 1. A microfluidic device for microbial co-cultivation.

(A) Compartmentalized co-cultures enable detection of symbiotic relations among community members. (B) Schematic design of the microfluidic device. (C) A picture of the microfluidic device. (D) Droplets filling a large chamber in the microfluidic device.

Figure 2. Comparisons between experiments and calculations for cell distribution in droplets.

Calculations were based on the Poisson distribution. (A) Numbers of droplets carrying different numbers of cells. (B) Numbers of droplets carrying four different combinations of a two-strain system. (C) Numbers of droplets carrying eight different combinations of a three-strain system.

Figure 3. On-chip cultivation of a cross-feeding pair.

(A) A synthetic symbiotic system consisting of two cross-feeding amino acid auxotrophs. (B) A section of the large cultivation chamber illustrating a number of droplets carrying four combinations of the two-strain system. E. coli strain Y^- is labeled with yellow fluorescence (EYFP) and W^- with red fluorescence (mCherry). Left – before cultivation, Right – Pictures after 18-hour cultivation. (C) Comparison of four individual droplets from Panel (B).

Figure 4. Comparison of a fast growing pair (K-12 W^- and Y^-) and a slow growing pair (EcNR1 W^- and Y^-) on the same device.

(A) Three droplets carrying the pair of E. coli K-12 W^- expressing mCherry and Y^- (not labeled with fluorescence). Top panels – before cultivation. Bottom panels – after 18-hour cultivation. (B) Three droplets carrying the pair of E. coli EcNR1 W^- expressing GFP and K-12 Y^-. Top panels – before cultivation. Bottom panels – after 18-hour cultivation.

Figure 5. On-chip cultivation of a triplet system.

(A) A synthetic system of three amino acid auxotrophs (W^-, Y^-, and S^-). Only W^- and Y^- forms a symbiotic relationship. (B) Eight droplets illustrating all the combinations of the triplet system. Top panels are pictures taken before the cultivation and bottom panels are at 18 hours.