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Droplet microfluidics has recently evolved as a prominent platform for high-throughput experimentation for various research fields including microbiology. Key features of droplet microfluidics, like compartmentalization, miniaturization, and parallelization, have enabled many possibilities for microbiology including cultivation of microorganisms at a single-cell level, study of microbial interactions in a community, detection and analysis of microbial products, and screening of extensive microbial libraries with ultrahigh-throughput and minimal reagent consumptions. In this book chapter, we present several aspects and applications of droplet microfluidics for its implementation in various fields of microbial biotechnology. Recent advances in the cultivation of microorganisms in droplets including methods for isolation and domestication of rare microbes are reviewed. Similarly, a comparison of different detection and analysis techniques for microbial activities is summarized. Finally, several microbial applications are discussed with a focus on exploring new antimicrobials and high-throughput enzyme activity screening. We aim to highlight the advantages, limitations, and current developments in droplet microfluidics for microbial biotechnology while envisioning its enormous potential applications in the future., (© 2020. Springer Nature Switzerland AG.)

Background: Cell-based experimentation in microfluidic droplets is becoming increasingly popular among biotechnologists and microbiologists, since inherent characteristics of droplets allow high throughput at low cost and space investment. The range of applications for droplet assays is expanding from single cell analysis toward complex cell-cell incubation and interaction studies. As a result of cellular metabolism in these setups, relevant physicochemical alterations frequently occur before functional assays are conducted. However, to use droplets as truly miniaturized bioreactors, parameters like pH and oxygen availability should be controlled similar to large-scale fermentation to ensure reliable research., Results: Here, we introduce a comprehensive strategy to monitor and control pH for large droplet populations during long-term incubation. We show the correlation of fluorescence intensity of 6-carboxyfluorescein and pH in single droplets and entire droplet populations. By taking advantage of inter-droplet transport of pH-mediating molecules, the average pH value of several million droplets is simultaneously adjusted in an a priori defined direction. To demonstrate the need of pH control in practice, we compared the fermentation profiles of two E. coli strains, a K12-strain and a B-strain, in unbuffered medium with 5 g/L glucose for standard 1 L bioreactors and 180 pL droplets. In both fermentation formats, the commonly used B-strain E. coli BL21 is able to consume glucose until depletion and prevent a pH drop, while the growth of the K12-strain E. coli MG1655 is soon inhibited by a low pH caused by its own high acetate production. By regulating the pH during fermentation in droplets with our suggested strategy, we were able to prevent the growth arrest of E. coli MG1655 and obtained an equally high biomass yield as with E. coli BL21., Conclusion: We demonstrated a comparable success of pH monitoring and regulation for fermentations in 1 L scale and 180 pL scale for two E. coli strains. This strategy has the potential to improve cell-based experiments for various microbial systems in microfluidic droplets and opens the possibility for new functional assay designs.

The natural bacterial diversity is regarded as a treasure trove for natural products. However, accessing complex cell mixtures derived from environmental samples in standardized high-throughput screenings is challenging. Here, we present a droplet-based microfluidic platform for ultrahigh-throughput screenings able to directly harness the diversity of entire microbial communities. This platform combines extensive cultivation protocols in aqueous droplets starting from single cells or spores with modular detection methods for produced antimicrobial compounds. After long-term incubation for bacterial cell propagation and metabolite production, we implemented a setup for mass spectrometric analysis relying on direct electrospray ionization and injection of single droplets. Even in the presence of dense biomass we show robust detection of streptomycin on the single droplet level. Furthermore, we developed an ultrahigh-throughput screening based on a functional whole-cell assay by picoinjecting reporter cells into droplets. Depending on the survival of reporter cells, droplets were selected for the isolation of producing bacteria, which we demonstrated for a microbial soil community. The established ultrahigh-throughput screening for producers of antibiotics in miniaturized bioreactors in which diverse cell mixtures can be screened on the single cell level is a promising approach to find novel antimicrobial scaffolds.

In order to leverage the immense potential of droplet microfluidics, it is necessary to simplify the process of chip design and fabrication. While polydimethylsiloxane (PDMS) replica molding has greatly revolutionized the chip-production process, its dependence on 2D-limited photolithography has restricted the design possibilities, as well as further dissemination of microfluidics to non-specialized labs. To break free from these restrictions while keeping fabrication straighforward, we introduce an approach to produce complex multi-height (3D) droplet microfluidic glass molds and subsequent chip production by PDMS replica molding. The glass molds are fabricated with sub-micrometric resolution using femtosecond laser machining technology, which allows directly realizing designs with multiple levels or even continuously changing heights. The presented technique significantly expands the experimental capabilities of the droplet microfluidic chip. It allows direct fabrication of multilevel structures such as droplet traps for prolonged observation and optical fiber integration for fluorescence detection. Furthermore, the fabrication of novel structures based on sloped channels (ramps) enables improved droplet reinjection and picoinjection or even a multi-parallelized drop generator based on gradients of confinement. The fabrication of these and other 3D-features is currently only available at such resolution by the presented strategy. Together with the simplicity of PDMS replica molding, this provides an accessible solution for both specialized and non-specialized labs to customize microfluidic experimentation and expand their possibilities. [ABSTRACT FROM AUTHOR]

The majority of today's antimicrobial therapeutics is derived from secondary metabolites produced by Actinobacteria. While it is generally assumed that less than 1% of Actinobacteria species from soil habitats have been cultivated so far, classic screening approaches fail to supply new substances, often due to limited throughput and frequent rediscovery of already known strains. To overcome these restrictions, we implement high-throughput cultivation of soil-derived Actinobacteria in microfluidic pL-droplets by generating more than 600 000 pure cultures per hour from a spore suspension that can subsequently be incubated for days to weeks. Moreover, we introduce triggered imaging with real-time image-based droplet classification as a novel universal method for pL-droplet sorting. Growth-dependent droplet sorting at frequencies above 100 Hz is performed for label-free enrichment and extraction of microcultures. The combination of both cultivation of Actinobacteria in pL-droplets and real-time detection of growing Actinobacteria has great potential in screening for yet unknown species as well as their undiscovered natural products. [ABSTRACT FROM AUTHOR]

Droplet microfluidics offers a unique opportunity for ultrahigh-throughput experimentation with minimal sample consumption and thus has obtained increasing attention, particularly for biological applications. Detection and measurements of analytes or biomarkers in tiny droplets are essential for proper analysis of biological and chemical assays like single-cell studies, cytometry, nucleic acid detection, protein quantification, environmental monitoring, drug discovery, and point-of-care diagnostics. Current detection setups widely use microscopes as a central device and other free-space optical components. However, microscopic setups are bulky, complicated, not flexible, and expensive. Furthermore, they require precise optical alignments, specialized optical and technical knowledge, and cumbersome maintenance. The establishment of efficient, simple, and cheap detection methods is one of the bottlenecks for adopting microfluidic strategies for diverse bioanalytical applications and widespread laboratory use. Together with great advances in optofluidic components, the integration of optical fibers as a light guiding medium into microfluidic chips has recently revolutionized analytical possibilities. Optical fibers embedded in a microfluidic platform provide a simpler, more flexible, lower-cost, and sensitive setup for the detection of several parameters from biological and chemical samples and enable widespread, hands-on application much beyond thriving point-of-care developments. In this review, we examine recent developments in droplet microfluidic systems using optical fiber as a light guiding medium, primarily focusing on different optical detection methods such as fluorescence, absorbance, light scattering, and Raman scattering and the potential applications in biochemistry and biotechnology that are and will be arising from this. [ABSTRACT FROM AUTHOR]

Microfluidic emulsion-based droplet systems have a great potential for inexpensive ultrahigh-throughput experimentation. Yet, picking and upscaling single unique picoliter-sized droplets of interest out of million others for deeper analysis is still a fundamental limitation. In order to overcome this missing gap, we present a system in which droplets of interest are collected into a collection chamber (DropLot) in high throughput and then slowly redirected to an agar surface or microtiter plate via a capillary tube passing through an optical sensor before exiting. The signal of each droplet triggers a positioning algorithm that ultimately places the flowing droplet at a defined position on a Petri dish or microtiter plate. Results indicate effective isolation of single droplets giving rise to colonies on agar surface with a recovery rate of over 93%. The possibility to isolate individual droplets provides a critical feature for interfacing droplet microfluidics with standard laboratory analysis and processing. [ABSTRACT FROM AUTHOR]