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. 2018 Feb 14;8:3000. doi: 10.1038/s41598-018-20877-1

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Buoyancy separation of whole cell biocatalysts according to their growth rate. (a) The specific growth rate of E. coli EcNR1 variants on d-lactose was determined. Cells from two slow- (G3, A11) and two fast-growing (C9, B3) variants were expanded to microcolonies in nLRs. In the presence of H₂O₂, the majority of microcolonies formed from the fast-growing cells produced ascending nLRs (C9: 139 of 171; B3: 156 of 170) whereas the slow-growing cells did not (G3: 0 of 157; A11: 1 of 150). One-way ANOVA indicated a statistically significant difference among the different strains for both cells per colony (F(3,745) = 276.80, p < 0.00001, α = 0.01) and separation (F(3,32) = 373.25, p < 0.00001, α = 0.01). Data shown as mean ± SD, n = 9 with 14 to 21 nLRs per experiment. (b) Sectional drawing of the device used for separation (see also Supplementary Fig. 8). (c) The nLRs containing slow-growing strains were incubated in approx. 500-fold excess together with fast-growing strains until microcolonies had formed. Then, 1.73 × 10⁷ nLRs (with a total of 9.72 × 10⁶ microcolonies) were buoyancy-separated using the device shown in b. The nLRs containing the fast-growing variants B3 and C9 also carried a fluorescence label which allowed identifying the genotype by epifluorescence microscopy before (upper picture) and after (lower picture) separation (GFP filter set, see Supplementary Methods). A sample of 20,000 nLRs of the initially prepared total population and the entire buoyancy-positive top fraction (22,585 nLRs) were analysed by large particle sorting to determine the enrichment factor. Scale bar: 400 µm. (d) A total of approx. 16,000 cells representing 18 strain variants with different growth rates were distributed over approx. 50,000 nLRs and subjected to buoyancy separation after incubation. Specific genotypes in a subset (48 clones of each subset) of the total population before and after separation were determined (upper graph) and used for the calculation of the variant abundance (variants after separation x variants before separation⁻¹ − 1, lower graph). The triangle indicates the specific growth rate of unmodified E. coli EcNR1. N/A: Samples with more than one genotype per nLR (e.g. nLRs that had received two or more library cells during inoculation).