Microdroplet-Enabled Highly Parallel Co-Cultivation of Microbial Communities
Citation: Park J, Kerner A, Burns MA, Lin XN (
Microdroplet-Enabled Highly Parallel Co-Cultivation of Microbial Communities
Jihyang Park 0
Alissa Kerner 0
Mark A. Burns 0
Xiaoxia Nina Lin 0
Alfredo Herrera-Estrella, Cinvestav, Mexico
0 1 Department of Chemical Engineering, University of Michigan , Ann Arbor , Michigan, United States of America, 2 Department of Biomedical Engineering, University of Michigan , Ann Arbor , Michigan, United States of America, 3 Center for Computational Medicine and Bioinformatics, University of Michigan , Ann Arbor, Michigan , United States of America
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 mm65 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.
-
Competing Interests: RainDance Technologies provided the PFPE-PEG block copolymer surfactant used in this work and permitted its use solely for the
purpose of scientific research at the University of Michigan only under the direction of Dr. Xiaoxia Lin. This does not alter the authors adherence to all the PLoS
ONE policies on sharing data and materials.
In nature, most microbes live in synergistic communities as a
way to adapt to and thrive in various environments, such as the
ocean[1,2], soil[3,4], and higher organisms as hosts[5,6]. These
microbial communities play important roles in a wide spectrum of
ecosystems and form diverse interactions among community
members and with their surroundings[7]. For example, the human
body is a representative host for natural microbial communities:
over 100 trillion bacteria are estimated to be present in the human
gut[8], more than 600 microbial species are known to inhabit the
human oral cavity[9], and over 100 different bacterial 16S rRNA
are present on human skin[10]. These microbes are believed to be
closely related to human health[11]. For instance, the gut
microbiota is known to contribute to digestion of nutrients[12],
stimulation of immunity[13] and protection of the host from
inflammatory diseases[14]. Despite their ubiquitousness and
apparent significance, our understanding of these microbial
communities remains very limited, largely owing to their inherent
complexity and the difficulty in laboratory cultivation of most of
the microbes.
The majority of existing microbial species, estimated to be in
the millions[15], have not been cultured in the laboratory[16],
which severely limits the extent to which they can be
characterized and further studied. One important reason behind this
unculturability is that conventional laboratory cultivation is
aimed at pure cultures of individual species, while in nature, the
survival and growth of microorganisms are largely associated with
their interactions with other members of the community they live
in[7,16,17,18]. These interactions are mediated by various
molecules such as secondary metabolites, quorum sensing
molecules, and peptides[19,20,21]. Accordingly, researchers have
attempted to develop alternative cultivation techniques that allow
interactions among microbes[16,22,23,24,25]. For example,
Kaeberlein et al. successfully isolated and cultured previously
uncultivated marine microorganisms by using a multi-chamber
set-up which allowed the diffusion of small molecules through
membranes[16].
Recent years have seen the increasing application of
microfluidics, a powerful technological platform featuring small-scale
and rapid operations, to cell cultivation and subsequent analysis.
In particular, microfluidic compartmentalization has been widely
utilized. For example, microwells have been used to confine and
culture various microorganisms[26,27], including bacteria of
which the growth was quorum-sensing dependent[28].
Microfluidically generated droplets represent another strategy for
creating localized environments for di (...truncated)