High-throughput detection of ethanol-producing cyanobacteria in a microdroplet platform

Abstract

Ethanol production by microorganisms is an important renewable energy source. Most processes involve fermentation of sugars from plant feedstock, but there is increasing interest in direct ethanol production by photosynthetic organisms. To facilitate this, a high-throughput screening technique for the detection of ethanol is required. Here, a method for the quantitative detection of ethanol in a microdroplet-based platform is described that can be used for screening cyanobacterial strains to identify those with the highest ethanol productivity levels. The detection of ethanol by enzymatic assay was optimized both in bulk and in microdroplets. In parallel, the encapsulation of engineered ethanol-producing cyanobacteria in microdroplets and their growth dynamics in microdroplet reservoirs were demonstrated. The combination of modular microdroplet operations including droplet generation for cyanobacteria encapsulation, droplet re-injection and pico-injection, and laser-induced fluorescence, were used to create this new platform to screen genetically engineered strains of cyanobacteria with different levels of ethanol production.

Keywords: biofuels; cyanobacteria; fluorescence; microdroplets.

Figure 1.

(a) Flowchart specifying the steps involved in the study. (b) Schematic of each microdroplet operation; from left to right: microdroplet formation for cell encapsulation, pico-injection of assay components in pre-formed droplets, fluorescence detection after ethanol conversion into RF. (c) Bright-field images corresponding to each step involved in the process.

Figure 2.

(a) Enzymatic reaction assay scheme; AOX: alcohol oxidase, HRP: horseradish peroxidase, AR: Amplex Red. (b) Ethanol assay calibration; standard ethanol solution absorbance (squares). (c) Ethanol production rate (squares) versus microalgae growth rate (triangles). (Online version in colour.)

Figure 3.

Bright-field images of (a) microdroplets after cell encapsulation, diameter = 90 µm. (b) Re-injection of microdroplets in a pico-injection device after 24 h incubation. (c) A re-injected microdroplet being pico-injected with the assay components for ethanol detection. (d) Microdroplets after pico-injection, diameter = 110 µm. (e) Histogram showing the number of cells per droplet distribution after encapsulation by using an 80 × 75 µm (w × h) flow-focusing microfluidic chip with flow rates of 2000 and 250 µl h⁻¹ for the oil and for the cell suspension, respectively. Discontinuous curve showing the Poisson curve corresponding to a 90 µm droplet (380 pl) for a cell concentration when droplet formation of 2.5 × 10⁶ cells ml⁻¹. (f) Histogram showing the fluorescence intensity distribution of the microdroplets after detection. In orange, pre-formed droplets just containing the culture media (BG11); in blue, the fluorescence pattern obtained after encapsulating the wild-type strain; and in green, the two-populations profile obtained when encapsulating the ethanol-producing strain.