Figure 2

Detection of antimicrobial substances in droplet supernatant by LCMS and inhibition assays. (a) Sketch of droplet pooling. The aqueous phase of ~ 1 × 10⁷ droplets occupied to 100% with microcolonies (λ ~ 10) is pooled and the biomass removed. (b) Extracted ion chromatogram at m/z = 503.2574 ± 0.0025 [M + H]⁺ for the reference substance streptothricin F (top panel). The lower panel displays for the same mass charge ratio the extracted ion chromatogram detected for a droplet supernatant, which was derived from a droplet population containing S. noursei. Streptothricin F is one of the four compounds which constitute nourseothricin, an aminoglycoside antibiotic. (c) Inhibition assay with the reporter strain B. subtilis and droplet supernatant derived from a droplet population containing S. noursei. Growth of B. subtilis was observed by monitoring the increase in red fluorescence, as the gene for a red fluorescent protein mKate was constitutively expressed. Droplet supernatant and controls were mixed in a volume ratio of 1:1 with a B. subtilis culture of OD₆₀₀ 1. As control a B. subtilis culture was incubated with fresh medium, which was used upon droplet generation. To mimick the nutrient consumption of S. noursei in droplets, PBS was mixed with the reporter as a second positive control. As negative control the reporter strain was incubated with medium containing 10 μg/ml chlortetracycline (CTC). (d) Following the same principle as described for (c) the droplet supernatant of S. collinus was tested for inhibition with the reporter strain E. coli.