With regard to biofuel feedstocks, microalgae have competitive advantages such as faster growth, higher lipid yield, and less competition with food supply and land usage compared to oil-producing crops such as corn, soybean, and sugarcane that are currently being used for ethanol, biodiesel, or second-generation fuel production. Despite this tremendous potential, microalgal biofuel production is neither economically competitive nor fully sustainable, and significant improvements are required for commercial viability.

Developing microalgal strains with faster growth rates and/or higher lipid content through genetic improvement and metabolic engineering is an important component to overcoming existing barriers to commercial development. … Successful screening for such mutants requires single-cell resolution measurement of the attributes of interest from large mutant populations, as each member of the population potentially has a unique profile. However, conventional screening where diluted pools of cells are cultured on media plates and transferred into separate microplate wells for the characterization of individuals, is too laborious and time-consuming to employ at large scale.

… Here, we present a high-throughput droplet microfluidics-based screening platform that allows for analyzing growth and lipid content from microalgal cells, followed by selective retrieval and off-chip analysis of samples showing the enhanced traits.

—Kim et al.

Illustration of the two-module high-throughput droplet microfluidics-based microalgae screening platform. (a) Droplet generation/culture module. (b) Droplet staining/analysis/sorting module. Kim et al. Click to enlarge.

The screening platform is composed of two interconnected micromodules: a droplet generation/culture module and a droplet staining/analysis/sorting module. In the droplet generation/culture module, single microalgal cells suspended in culture media are individually encapsulated into droplets through a T-junction droplet generator. The droplets then flow into a downstream culture chamber where they are incubated for 1.5–4 days to achieve growth inside the droplets

An array of pillars separated by 40-μm gaps was utilized along the side walls of the culture chamber to allow continuous carrier oil flow while all droplets remained trapped inside, resulting in a highly packed droplet array inside the culture chamber.

The incubated droplets are then transferred to the droplet staining/analysis/sorting module through a tubing connection. This module has three functional parts: an on-chip droplet staining region, a droplet incubation region, and a droplet analysis/sorting region.

Growth and lipid content are quantified through an integrated optical detection system while the incubated droplets flow through an optical detection channel in the analysis/sorting region.

This is the first microsystem that allows both lipid content analysis and growth rate measurement at high throughput, whereas previous work could only do one or the other.

—senior author Arum Han of Texas A&M University

Scientists are work to identify algal strains that can reproduce faster and produce more lipid per cell. This summer, ExxonMobil announced the discovery of a strain with a single genetic modification that allows for twice as much lipid production per cell.This is only a step in the right direction, as thousands of genes hold potential for further improving both traits.

With today’s gene-editing technologies, modifying algal genes can be relatively straightforward; however, identifying which genes to target is time-consuming and costly. Exposing an algal culture to a mutagen yields millions of unique, potentially improved algal cells that must each be tested for expression of a desired trait, such as increased lipid production. Mutated genes can then be identified through whole-genome sequencing.

The important thing is to develop a tool that can screen millions of cells in a much shorter time frame and a smaller space. In a chip housing millions of droplets of cells, each droplet is like a flask or a bioreactor, and that’s how we can get results faster from just a tiny chip.

—Shih-Chi Hsu, author and BTI post-doc

The researchers first validated the chip system with algae known to grow faster or slower, or produce more or less lipid. They then screened 200,000 chemically mutated cells, identifying six mutants with both faster growth and higher lipid content. The screening, done on-chip, uses fluorescence detection of chlorophyll, representing total cell mass, and BODIPY, a fluorescent molecule that binds to lipids. All mutants with potential for improved growth or lipid production were recovered and verified off-chip.

While the results of this study are promising, 200,000 is still a low number of mutants compared to what is needed to find a super algal strain.

The most extraordinary variants will be found in one in a million, or ten million, so the throughput needs to be accelerated.

—BTI President David Stern

Tools for improving throughput are already in development, including larger chips that can screen millions of droplets in one experiment.

Resources

• Kim HS, Hsu S-C, Han S-I, et al. (2017) “High-throughput droplet microfluidics screening platform for selecting fast-growing and high lipid-producing microalgae from a mutant library. Plant Direct 00:1–13 doi: 10.1002/pld3.11