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The application of microfluidic techniques in experimental and environmental studies is a rapidly emerging field. Water-in-oil microdroplets can serve readily as controllable micro-vessels for studies that require spatial structure. In many applications, it is useful to monitor cell growth without breaking or disrupting the microdroplets. To this end, optical reporters based on color, fluorescence, or luminescence have been developed. However, optical reporters suffer from limitations when used in microdroplets such as inaccurate readings due to strong background interference or limited sensitivity during early growth stages. In addition, optical detection is typically not amenable to filamentous or biofilm-producing organisms that have significant nonlinear changes in opacity and light scattering during growth. To overcome such limitations, we show that volatile methyl halide gases produced by reporter cells expressing a methyl halide transferase (MHT) can serve as an alternative nonoptical detection approach suitable for microdroplets. In this study, an MHT-labeled Streptomyces venezuelae reporter strain was constructed and characterized. Protocols were established for the encapsulation and incubation of S. venezuelae in microdroplets. We observed the complete life cycle for S. venezuelae including the vegetative expansion of mycelia, mycelial fragmentation, and late-stage sporulation. Methyl bromide (MeBr) production was detected by gas chromatography-mass spectrometry (GC-MS) from S. venezuelae gas reporters incubated in either liquid suspension or microdroplets and used to quantitatively estimate bacterial density. Overall, using MeBr production as a means of quantifying bacterial growth provided a 100- to 1,000-fold increase in sensitivity over optical or fluorescence measurements of a comparable reporter strain expressing fluorescent proteins. IMPORTANCE Quantitative measurement of bacterial growth in microdroplets in situ is desirable but challenging. Current optical reporter systems suffer from limitations when applied to filamentous or biofilm-producing organisms. In this study, we demonstrate that volatile methyl halide gas production can serve as a quantitative nonoptical growth assay for filamentous bacteria encapsulated in microdroplets. We constructed an S. venezuelae gas reporter strain and observed a complete life cycle for encapsulated S. venezuelae in microdroplets, establishing microdroplets as an alternative growth environment for Streptomyces spp. that can provide spatial structure. We detected MeBr production from both liquid suspension and microdroplets with a 100- to 1,000-fold increase in signal-to-noise ratio compared to optical assays. Importantly, we could reliably detect bacteria with densities down to 106 CFU/mL. The combination of quantitative gas reporting and microdroplet systems provides a valuable approach to studying fastidious organisms that require spatial structure such as those found typically in soils.

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Spatial structure within microbial communities can provide nearly limitless opportunities for social interactions and are an important driver for evolution. As metabolites are often molecular signals, metabolite diffusion within microbial communities can affect the composition and dynamics of the community in a manner that can be challenging to deconstruct. We used encapsulation of a synthetic microbial community within microdroplets to investigate the effects of spatial structure and metabolite diffusion on population dynamics and to examine the effects of cheating by one member of the community. The synthetic community was comprised of three strains: a 'Producer' that makes the diffusible quorum sensing molecule ( N -(3-Oxododecanoyl)-L-homoserine lactone, C12-oxo-HSL) or AHL; a 'Receiver' that is killed by AHL and a Non-Producer or 'cheater' that benefits from the extinction of the Receivers, but without the costs associated with the AHL synthesis. We demonstrate that despite rapid diffusion of AHL between microdroplets, the spatial structure imposed by the microdroplets allow a more efficient but transient enrichment of more rare and slower growing 'Producer' subpopulations. Eventually, the Non-Producer population drove the Producers to extinction. By including fluorescence-activated microdroplet sorting and providing sustained competition by the Receiver strain, we demonstrate a strategy for indirect enrichment of a rare and unlabeled Producer. The ability to screen and enrich metabolite Producers from a much larger population under conditions of rapid diffusion provides an important framework for the development of applications in synthetic ecology and biotechnology.

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Microdroplet emulsions allow investigators to build controllable microenvironments for applications in experimental evolution and synthetic ecology. We designed a microfluidic platform that uses highly homogenous microdroplets to enable these experiments. We also present a step-by-step protocol for the rapid production of highly homogeneous microdroplets suitable for experimental evolution. We also describe protocols for the propagation and serial passage of microbial populations across a range of selection schemes and potential spatial structures. For complete details on the use and execution of this protocol, please refer to Seo et al. (2021).

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In vitro experimental evolution of pathogens to antibiotics is commonly used for the identification of clinical biomarkers associated with antibiotic resistance. Microdroplet emulsions allow exquisite control of spatial structure, species complexity, and selection microenvironments for such studies. We investigated the use of monodisperse microdroplets in experimental evolution. Using Escherichia coli adaptation to doxycycline, we examined how changes in environmental conditions such as droplet size, starting lambda value, selection strength, and incubation method affected evolutionary outcomes. We also examined the extent to which emulsions could reveal potentially new evolutionary trajectories and dynamics associated with antimicrobial resistance. Interestingly, we identified both expected and unexpected evolutionary trajectories including large-scale chromosomal rearrangements and amplification that were not observed in suspension culture methods. As microdroplet emulsions are well-suited for automation and provide exceptional control of conditions, they can provide a high-throughput approach for biomarker identification as well as preclinical evaluation of lead compounds.

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