Microfluidic high-throughput culturing of single cells for selection based on extracellular metabolite production or consumption

Wang, Benjamin; Ghaderi, Adel; Zhou, Hang; Agresti, Jeremy J.; Weitz, David A.; Fink, Gerald R.; Stephanopoulos, Gregory

doi:10.1038/nbt.2857

Cited by 311 publications

(259 citation statements)

References 35 publications

(34 reference statements)

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“…New methods allow increasingly high-throughput culturing of individual microbial strains and can even select variant individuals from a single species with particular functional abilities (55). Hand-selected isolates might potentially be combined into designer soil inocula, but the interactions between these isolates in situ will be difficult to predict, as will the consistency of their functions, phenotypes, and mobile genomes.…”

Section: Resultsmentioning

confidence: 99%

A Diverse Soil Microbiome Degrades More Crude Oil than Specialized Bacterial Assemblages Obtained in Culture

Bell

Stefani

Abram

et al. 2016

Appl Environ Microbiol

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Soil microbiome modification may alter system function, which may enhance processes like bioremediation. In this study, we filled microcosms with gamma-irradiated soil that was reinoculated with the initial soil or cultivated bacterial subsets obtained on regular media (REG-M) or media containing crude oil (CO-M). We allowed 8 weeks for microbiome stabilization, added crude oil and monoammonium phosphate, incubated the microcosms for another 6 weeks, and then measured the biodegradation of crude oil components, bacterial taxonomy, and functional gene composition. We hypothesized that the biodegradation of targeted crude oil components would be enhanced by limiting the microbial taxa competing for resources and by specifically selecting bacteria involved in crude oil biodegradation (i.e., CO-M). Postincubation, large differences in taxonomy and functional gene composition between the three microbiome types remained, indicating that purposeful soil microbiome structuring is feasible. Although phylum-level bacterial taxonomy was constrained, operational taxonomic unit composition varied between microbiome types. Contrary to our hypothesis, the biodegradation of C 10 to C 50 hydrocarbons was highest when the original microbiome was reinoculated, despite a higher relative abundance of alkane hydroxylase genes in the CO-M microbiomes and of carbon-processing genes in the REG-M microbiomes. Despite increases in the relative abundances of genes potentially linked to hydrocarbon processing in cultivated subsets of the microbiome, reinoculation of the initial microbiome led to maximum biodegradation. IMPORTANCEIn this study, we show that it is possible to sustainably modify microbial assemblages in soil. This has implications for biotechnology, as modification of gut microbial assemblages has led to improved treatments for diseases like Clostridium difficile infection. Although the soil environment determined which major phylogenetic groups of bacteria would dominate the assemblage, we saw differences at lower levels of taxonomy and in functional gene composition (e.g., genes related to hydrocarbon degradation). Further studies are needed to determine the success of such an approach in nonsterile environments. Although the biodegradation of certain crude oil fractions was still the highest when we inoculated with the diverse initial microbiome, the possibility of discovering and establishing microbiomes that are more efficient in crude oil degradation is not precluded. O il production, oil spills, and the storage of oily wastes by the petroleum industry have led to massive releases of petroleum hydrocarbons into the environment, making them some of the most ubiquitous environmental pollutants (1). Conventional decontamination technologies such as excavation (i.e., dig and dump), incineration, and chemical treatment of contaminants are costly and may further disrupt disturbed ecosystems (2, 3). When effective, bioremediation is a cheaper and more sustainable approach to decontamination. Bioaugmentation, the additio...

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Section: Resultsmentioning

confidence: 99%

A Diverse Soil Microbiome Degrades More Crude Oil than Specialized Bacterial Assemblages Obtained in Culture

Bell

Stefani

Abram

et al. 2016

Appl Environ Microbiol

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“…If the droplet contains enough nutrients, cells can be cultured within droplets [34] (Figure 2). Interestingly, the gas exchange needed for culturing can be readily provided, typically by the use of fluorinated oils with high oxygen permeability or through the device itself.…”

Section: Cell Biology and Tissue Engineeringmentioning

confidence: 99%

“…On-chip lysis of cells within droplets can be chemically achieved using a pulsed laser microbeam, using electroporation or using the addition of lysis buffer to the droplets during encapsulation [37]. [34].…”

Section: Cell Biology and Tissue Engineeringmentioning

confidence: 99%

Droplet microfluidics: A tool for biology, chemistry and nanotechnology

Mashaghi

Abbaspourrad

Weitz

et al. 2016

TrAC Trends in Analytical Chemistry

310

188

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The ability to perform laboratory operations on small scales using miniaturized devices provides numerous benefits, including reduced quantities of reagents and waste as well as increased portability and controllability of assays. These operations can involve reaction components in the solution phase and as a result, their miniaturization can be accomplished through microfluidic approaches. One such approach, droplet microfluidics, provides a high-throughput platform for a wide range of assays and approaches in chemistry, biology and nanotechnology. We highlight recent advances in the application of droplet microfluidics in chipbased technologies, such as single-cell analysis tools, small-scale cell cultures, in-droplet chemical synthesis, high-throughput drug screening, and nanodevice fabrication.

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“…64 This allows for accurate microbial performance assessments and subsequent droplet sorting to collect microbes with desirable phenotypes typically at ∼1 kHz, and potentially up to 30 kHz. 65 These characteristics give FADS an access to various phenotypic screenings such as screening for cell culture 66 and single cell secretion level of proteins and metabolites, 6,67,68 that are not accessible to FACS as FACS assay is inherently limited to fluorescence within the cell or on the cell membrane. 69 Huang et al used droplet microfluidics to screen 10 5 to 10 6 of UV-irradiated S. cerevisiae variants with desired α-amylase secretion rates (Fig.…”

Section: Fluorescent Phenotypic Analysis In Dropletsmentioning

confidence: 99%