High-throughput screening of high lactic acid-producing Bacillus coagulans by droplet microfluidic based flow cytometry with fluorescence activated cell sorting

Abstract

A high-throughput screening system based on droplet microfluidic sorting was developed and employed for screening of high lactic acid-producing Bacillus coagulans. In this system, water-in-oil-in-water (W/O/W) droplets, which were ∼12 pL in volume were used as picoliter-reactors for lactic acid fermentation. A fluorescent sensor was developed and used for monitoring pH which indicated the production of lactic acid. After fermentation, fluorescence activated cell sorting was performed with high sensitivity and speed. Using this microfluidic high-throughput screening system, we found a mutant with a yield of 76 g L^-1 lactic acid which was 52% higher than its parent strain with a screening throughput exceeding 10⁶ clones per h.

Fig. 1. Schematic illustration of the proposed droplet microfluidic system. (A) The response mechanism of the pH fluorescent biosensor. (B) Design of chip and generation of W/O/W droplets. (C) Cell growing and producing of lactic acid and the response of the fluorescent. (D) Ultrahigh-throughput screening combined with FACS.

Fig. 2. Chip design and generation of W/O/W microdroplets. (A) Design and photographs of the one-step W/O/W microdroplet generator. Scale bar: 100 μm. (B) Microphotograph of W/O/W microdroplets. Scale bar: 100 μm. (C) The outer and inner diameters distribution of W/O/W microdroplets.

Fig. 3. Lactic acid assay in microdroplets. (A) Fluorescence imaging of microdroplets containing broth with a range of lactic acid, 8 g L⁻¹ (I), 11 g L⁻¹ (II), 12 g L⁻¹ (III), 14 g L⁻¹ (IV). Scale bar: 200 μm. (B) The corresponding average gray value derive from the fluorescence intensity of the microdroplets in the images of (A). (C) The microdroplets cultured for 10 hours showed difference in fluorescence. Microdroplets with relative high, low fluorescence intensity and the empty ones were illustrated by (iii), (ii), (i), respectively. Scale bar: 50 μm.

Fig. 4. High throughput screening of W/O/W microdroplets by FACS. (A) Alexa Fluor 430 – A against SSC-A (side scatter-area) log–log plots of the control group, which was not cultured. (B) Alexa Fluor 430 – A against SSC-A (side scatter-area) log–log plots of the samples after cultivation. The Q2 gating events was considered as the microdroplets with cells which were producing lactic acid. (C) Fluorescence frequency histograms derived from all the events of (A) (red) and (B) (blue). Three peaks (i), (ii), (iii) indicated the O/W single emulsion and fragments, W/O/W without cells and W/O/W with cells as shown in the fluorescence images. Scale bar: 50 μm. (D) and (E) Droplets before and after FACS sorting. It showed an obvious enrichment of droplets with strong fluorescence intensity. Scale bar: 100 μm (F) 10 mutants with high yield of lactic acid and three generations hereditary stability was obtained. The labels on the X-axis represented the name of 10 mutants, and the three column of each mutant represented the lactic acid production of each generation (G1, G2, G3). The highest yield of mutant E11 reached 76 g L⁻¹ (indicated by blue arrow), 52% higher than its mother strains.